Workflow for detection of ligands using nucleic acids

ABSTRACT

This application relates to methods for ligating oligonucleotides having complementarity to a target nucleic acid, and amplifying the ligated oligonucleotides, where ligation and amplification occur in the same reaction mixture.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a division of U.S. patent application Ser. No.16/656,839, filed Oct. 18, 2019; which is a continuation of U.S. patentapplication Ser. No. 14/955,386, filed Dec. 1, 2015 (now U.S. Pat. No.10,472,671); which is a continuation of U.S. patent application Ser. No.13/352,237, filed Jan. 17, 2012 (now abandoned); which claims priorityto U.S. Provisional Patent Application No. 61/433,475, filed Jan. 17,2011, the disclosures of all of which are hereby incorporated byreference in their entirety, as if set forth fully herein.

FIELD OF THE DISCLOSURE

This application relates to methods for ligating oligonucleotides havingcomplementarity to a target nucleic acid, and amplifying the ligatedoligonucleotides, where ligation and amplification occur in the samereaction mixture.

BACKGROUND OF THE DISCLOSURE

The correlation of gene and protein expression changes in biologicalsystems has been hampered by the need for separate sample handling andanalysis platforms for nucleic acids and proteins. In contrast to thesimple, rapid, and flexible workflow of quantitative PCR (qPCR) methods,which enable characterization of several classes of nucleic acidbiomarkers (e.g., DNA, mRNA, and microRNAs), protein analysis methodssuch as Western blotting are cumbersome, laborious, and much lessquantitative. Proximity Ligation Assays (PLAs) have been shown toeliminate some of these problems. However, improvements to PLAs aredesired by those of skill in the art.

Typical or conventional PLAs usually involve at least three or foursteps. The first step is typically the binding of first and secondprobes (e.g., antibody probes) to a ligand (e.g., a protein of interest)such that the probes are in close proximity to another. Each of theprobes typically contain an oligonucleotide. The oligonucleotides arebrought into proximity to one another with the binding of the probesand, in the second step, are then ligated to one another (e.g., theligation event). The ligated oligonucleotides may then be amplified anddetected to determine the presence of the ligand with a test sample(e.g., a biological sample). This step is typically accomplished byadding ligation components, such as ligase, adenosine triphosphate (ATP)and buffer-salt mixture, to the binding reaction. In the third step, theligase is typically then deactivated (e.g., by protease digestion) toprevent any further ligation of unbound oligonucleotides. In the fourthstep, the reaction mixture is transferred to a real-time polymerasechain reaction (PCR) mixture and the quantity of the amplified productdetermined by quantitative PCR (qPCR). As described below, it has beensurprisingly found that the third step (ligase digestion) may beeliminated, thereby allowing ligation and amplification to occur in thesame reaction mixture without inactivation of the ligase. These andother features and advantages of the methods described herein will beapparent to the skilled artisan from this disclosure.

SUMMARY OF THE DISCLOSURE

Provided herein are methods for ligating and amplifyingoligonucleotides. In some embodiments, the oligonucleotides are attachedto ligand-specific probes, and amplification of the oligonucleotidesindicates that the probes have bound a ligand in the sample. In oneembodiment, a method for ligating at least two oligonucleotides toproduce a ligated oligonucleotide and amplifying the ligatedoligonucleotide, wherein ligation and amplification occur in a singlereaction mixture (e.g., that may be considered undiluted) is provided.In some embodiments, a third oligonucleotide may be used to bridge theat least two oligonucleotides that are bound to the probes. In certainembodiments, the method may comprise detecting a ligand in a test sample(e.g., a biological sample) comprising contacting the protein with atleast a first and second probe, each probe having binding specificityfor the protein and being adjoined to at least one type ofoligonucleotide, the oligonucleotides on the first and second probes,respectively, being at least partially complementary to one another;ligating the oligonucleotides on the first and second probes to oneanother using a ligase to produce a target nucleic acid and amplifyingthe target nucleic acid; and, detecting the amplified target nucleicacid. For instance, the method may comprise detecting a protein in atest sample, the method comprising contacting the protein with at leasttwo probes having binding specificity therewith, each of the two agentscomprising at least one oligonucleotide; ligating the oligonucleotidesto produce a ligated oligonucleotide and amplifying the ligatedoligonucleotide in a single reaction mixture; and, detectingamplification of the ligated oligonucleotide. In some embodiments, oneor more of the probes is an antibody. In certain embodiments, at leastone of the oligonucleotides comprises at least three nucleotides. Someembodiments provide for the oligonucleotide being ligated using a smallfootprint ligase, which may be contacted with adenosine triphosphateprior to use. Any type of amplification procedure may be used such as,without limitation, polymerase chain reaction (PCR) (e.g., quantitativePCR (qPCR)). In some embodiments, it may be beneficial to inactivate theligase prior to amplification (e.g., using a protease). Otherembodiments of the methods described herein will be apparent to theskilled artisan from the disclosure provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings described beloware for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1. A schematic diagram of an exemplary typical or conventional PLAprocess.

FIG. 2. A schematic diagram of an exemplary improved PLA process (asdisclosed herein).

FIGS. 3A and 3B. A schematic diagram of an exemplary typical orconventional PLA workflow (FIG. 3A), and a schematic diagram of anexemplary improved PLA workflow (FIG. 3B), as disclosed herein.

FIGS. 4A and 4B. A schematic diagram of exemplary oligonucleotidesplints using asymmetrical (e.g., 3+6) (FIG. 4A) and symmetrical (e.g.,4+4) (FIG. 4B) oligonucleotide splints (“connectors”).

FIG. 5. Comparison of a typical or conventional PLA process (PLA1) andthe improved process (PLA2) (dCT data shown).

FIG. 6. Use of the improved PLA process with various target nucleicacids.

FIG. 7. Comparison of two different splint lengths at varyingconcentrations.

FIG. 8. Comparison of five different splint lengths.

FIG. 9. Comparison of T4 ligase to two different SF ligases (e.g., SFand DLxD).

DETAILED DESCRIPTION

Disclosed herein are methods for performing proximity ligation assays.In typical or conventional proximity ligation assay (PLA) processes(FIG. 1), a probe mix and sample are combined into a binding reaction.Following the binding reaction, the ligation reaction mixture is addedin order to carry out the ligation reaction. To prepare the ligationreaction mixture, a ligase and ligation buffer are diluted. Followingthe ligation reaction, the ligated product is stabilized by proteasedigestion; the protease is then inactivated (e.g, using heat). A portionof the ligated product is transferred to a real-time PCR reactionmixture, then placed on a PCR reaction vessel (e.g., plate) in a qPCRinstrument. Detection and quantification of the ligated product thenproceeds using standard techniques.

In one embodiment of the improved PLA process disclosed herein, a celllysate may be prepared and a ligation buffer added thereto. To thatmixture may then be added a proximity probe mixture, a ligase, and a PCRmixture (which may include, for example, a thermostable polymerase).This combined reaction mixture can then incubated for a suitable amountof time (e.g., one hour, 37° C.), the ligase optionally inactivated(e.g., using heat) and PCR performed directly on the mixture. Aschematic of an exemplary embodiment of the improved PLA process isillustrated in FIG. 2. As shown therein, the binding reaction is thesame as that shown in FIG. 1. However, in some embodiments of theimproved PLA processes, the ligase is added to the real-time PCR mixturewhich is then added directly to the binding reaction. In someembodiments, this reaction mixture is then deposited onto a reactionplate and then analyzed by a qPCR instrument. Detection andquantification of the ligated product then proceeds using standardtechniques.

In some embodiments, the lesser dilution factor provided thereby mayresult in a higher PLA probe concentration in the ligation reaction. Theincreased probe concentration may cause increased background signal,which may be minimized by using a short splint oligonucleotide atreduced concentration. For instance, a suitable splint oligonucleotidemay be 14 nucleotides in length (e.g., at least five nucleotides in the3′ end and at least nine nucleotides in the 5′ end; 5+9). To ensure theligation efficiency, a small footprint ligase (SFL) may be used. In someembodiments, to further simplify the ligation-PCR step, the typicaladdition of ATP to the ligation reaction may be omitted. Instead, onemay optionally use an ATP-enriched SFL (e.g., an SFL that is exposed toor contacted with an abundance or additional supply of ATP for someperiod of time). This enrichment step may be especially useful whenco-substrates for other ligases are used. Thus, the binding reaction maybe assembled by combining proximity probes and samples containing targetmolecule(s), and incubating the mixture such that binding between theprobes and the target molecule(s) occurs. In some embodiments, after thebinding reaction, a ligation-PCR mix (e.g., comprising a short splintoligo (e.g., an oligonucleotide that is at least 6 nucleotides, e.g., 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides)in length, an SFL, and standard real-time PCR components) may be added.In some other embodiments, the ligation reaction may then take place atroom-temperature, and the product amplified and quantitated by real-timePCR. In some embodiments, the ligase may be deactivated (e.g., usingheat). The ligated product may then be subjected to real-time PCRimmediately or following storage. Thus, the various embodiments of thenovel work flows for improved PLA processes as disclosed herein providereduced dilution factors which enable one to accomplish ligation and PCRin a single reaction mixture (e.g., or in a single step withoutintermediate/intervening steps). In some preferred embodiments, a shortsplint oligonucleotide and an SFL may also be used to control anyincreased background reactions. In additional embodiments, the SFL maybe pre-enriched using ATP.

Exemplary typical and improved PLA processes are further compared inFIGS. 3A and 3B. As shown therein, the typical processes include samplepreparation, a binding reaction, ligation, ligase inactivation using aprotease, protease inactivation (e.g., using heat), followed byreal-time PCR. To carry out the PCR step in typical PLA processes, aportion of the reaction mixture containing the inactivated ligase andprotease is usually transferred to a PCR plate, and the “PCR mix” (e.g.,containing primers, dNTPs, polymerase, and the like) added thereto. Asshown in FIG. 3B, the disclosed improved processes can eliminate the useof a protease and dilution of the reaction mixture prior to PCR. Asshown therein, the ligase may be inactivated using, for example, heat,and the resultant reaction mixture placed directly into a qPCRassay/instrument. Thus, simplified, improved PLA work flows can useentire binding reaction products in a real-time PCR assay (e.g., in amulti-plate well). This provides an improved work-flow and reduceddilution of the reaction mixture. As a result, in some embodiments ofthe improved PLA processes, the PCR reaction mixture contains a higherconcentration of the ligated product (e.g., the target nucleic acid). Insome embodiments, the improvement provided by the improved PLA processescan be measured as the dCT of the reaction (e.g., a 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more signal (dCT); see, for example, FIG. 4A and FIG. 4B).Sensitivity of the improved PLA processes as compared to typical PLAprocesses can also be observed (e.g., as fold-change; see, for example,FIG. 5).

The processes described herein provide for, in some embodiments,detecting a protein in a test sample, the methods comprising contactingthe protein with at least two probes having binding specificitytherewith, each of the two probes comprising at least oneoligonucleotide; ligating the oligonucleotides to produce a ligatedoligonucleotide; amplifying the ligated oligonucleotide in a singlereaction mixture; and, detecting amplification of the ligatedoligonucleotide. In some embodiments, a third oligonucleotide may alsobe used to bridge each of the oligonucleotides attached to each of theprobes. In some embodiments, one or more of the probes is an antibody.In certain embodiments, at least one of the oligonucleotides comprisesat least three nucleotides. Some embodiments provide for use of a SFL,which may optionally be contacted with adenosine triphosphate (ATP)prior to use, for ligation of the oligonucleotides. Any type ofamplification procedure may be used such as, without limitation,polymerase chain reaction (PCR) (e.g., quantitative PCR). In someembodiments, it may be beneficial to inactivate the ligase prior toamplification (e.g., using a protease, heat, or any other methods knownin the art). Other embodiments of the inventions described herein willbe apparent to the skilled artisan from the disclosure provided herein.

In some embodiments, a method for detecting a target in a sample isprovided where the method includes the steps of binding a first and asecond probe, each of which binds specifically to the target, whereineach of the probes comprises an oligonucleotide portion (or tail);ligating the first and second oligonucleotide tails thereby producing aligated oligonucleotide template; and, performing a polymerase chainreaction (PCR) of the oligonucleotide template across the first andsecond oligonucleotide tails to quantify the said template. In someembodiments, the ligation and PCR steps may be performed in the samereaction mixture. In other embodiments, the method may include binding afirst and a second probe, wherein each probe binds specifically to thetarget, each of the probes comprise a oligonucleotide tail; ligating theoligonucleotide tails to produce a ligated oligonucleotide template andamplifying the template by PCR in a single step to produce an amplifiedtemplate; and, quantitating the amplified template. The probes maycomprise antibodies which specifically bind to the target. Theoligonucleotides may be ligated using a splint oligonucleotide (e.g.,splint oligos of at least 6 nucleotides in length having 3′ and 5′overhangs of, for example, 9+9, 9+8, 9+7, 9+6, 9+5, 8+8, 5+3, 4+7, 3+3nucleotides, or any other possible variations in length or symmetry ascontemplated and described in further detail below). In someembodiments, the ligase may be pre-enriched using ATP and/or inactivatedusing, for example, one or more proteases and/or heat. The amplifiedtemplate may be quantified by any suitable method including, forexample, real-time PCR (e.g., a TaqMan® assay or a molecular beaconassay).

The improved processes disclosed and/or exemplified herein providereduced work times from process start time to collection of results(e.g., faster), reduced hands-on time (e.g., simpler and cheaper),reduced lab plasticware usage (e.g., cheaper and more environmentallysound (“greener”)), and increased signals and sensitivities (e.g., moresensitive). In some embodiments, these improved processes providesimplified work flows by combining ligation and PCR steps, reduceddilution factors from the binding step to the ligation step, reducedbinding probe concentrations to enable reduced dilution factors, use ofshorter connector oligonucleotides (e.g., as few as 6 nucleotides inlength) to control background signals, use of lower connectoroligonucleotide concentrations to control background signals, use of SFligases to enable use of shorter connector oligonucleotide lengths,ATP-enriched SF ligase purification schemes to omit ATP in ligation-PCRstep, and/or enabling use of the entire reaction volume to improve PLAsignal and sensitivity.

The methods described herein are particularly useful in that the samemay be used with various systems for detecting proteins. Exemplary ofsuch systems include, for example, TaqMan® Protein Assays. TaqMan®Protein Assays are an adapted form of PLA™, a proximity ligation assaytechnology that combines antibody-protein binding with detection of thereporter nucleic acid by real-time PCR. Applied Biosystems has optimizedthis technique for use with crude cell and tissue lysates and combinedit with TaqMan® chemistry to create a highly sensitive and specificprocess for measuring protein expression in small samples. Assays havebeen developed for the detection of OCT3/4, NANOG, SOX2, and LIN28 inhuman embryonic stem cells, as well as ICAM1 and CSTB to measurerelative quantification in human cells. The basic steps of such assaysinclude binding of a protein target by paired assay probes, ligation ofthe oligonucleotides by a DNA ligase, and amplification of the ligationproduct by TaqMan® real-time PCR assay. The probes used in the firststep are typically target-specific antibodies conjugated tooligonucleotides through a biotin-streptavidin (SA) linkage. Eacholigonucleotide in the pair presents a 5′ or 3′ end that are broughtinto proximity when the assay probes bind to two different epitopes onthe target protein. The substrate for the ligase is typically a bridgestructure formed by hybridization of a third oligonucleotide to theoligonucleotide ends of the assay probe pair. This structure formspreferentially when the assay probes are in proximity to each other. Theligation product typically serves the template in the TaqMan® real-timePCR assay. The systems may be used to, for example, to perform proteinanalysis on small samples (e.g., stem cells, germ cell tumors),correlate and/or validate results from RNA and protein quantitation,analyze post-translational modifications, validate siRNA-induced genesilencing, and/or validate gene transfection/transduction experiments.Data generated using these systems may be analyzed using software suchas, for instance, ProteinAssist™ software package (Applied Biosystems™).

To more clearly and concisely describe and point out the subject matterof the present disclosure, the following definitions are provided forspecific terms, which are used in the following description and theappended claims. Throughout the specification, exemplification ofspecific terms should be considered as non-limiting examples.

As used herein the terms “nucleotide” or “nucleotide base” refer to anucleoside phosphate. It includes, but is not limited to, a naturalnucleotide, a synthetic nucleotide, a modified nucleotide, or asurrogate replacement moiety or universal nucleotide (e.g., inosine).The nucleoside phosphate may be a nucleoside monophosphate, a nucleosidediphosphate or a nucleoside triphosphate. A “nucleotide” refers to anucleotide, nucleoside or analog thereof. Optionally, the nucleotide isan N- or C-glycoside of a purine or pyrimidine base. (e.g.,deoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleosidecontaining D-ribose). Examples of other analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides. Nucleotide basesusually have a substituted or unsubstituted parent aromatic ring orrings. In certain embodiments, the aromatic ring or rings contain atleast one nitrogen atom. In certain embodiments, the nucleotide base iscapable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with anappropriately complementary nucleotide base. Exemplary nucleotide basesand analogs thereof include, but are not limited to, purines such as2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine,N6-Δ2-isopentenyladenine (6iA), N6-Δ2-isopentenyl-2-methylthioadenine(2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine(dmG), 7-methylguanine (7 mG), 2-thiopyrimidine, 6-thioguanine (6sG)hypoxanthine and O6-methylguanine; 7-deaza-purines such as7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidinessuch as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),4-thiothymine (4sT), 5,6-dihydrothymine, 04-methylthymine, uracil (U),4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indolessuch as nitroindole and 4-methylindole; pyrroles such as nitropyrrole;nebularine; base (Y); etc. In certain embodiments, nucleotide bases areuniversal nucleotide bases. Additional exemplary nucleotide bases can befound, e.g., in Fasman, 1989, Practical Handbook of Biochemistry andMolecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., and thereferences cited therein. A “universal base”, as used herein, is a basethat is complementary to more than one other base. Fully universal basescan pair with any of the bases typically found in naturally occurringnucleic acids. The base need not be equally capable of pairing with eachof the naturally occurring bases. Alternatively, the universal base maypair only or selectively with two or more bases but not all bases.Optionally the universal base pairs only or selectively with purines, oralternatively with pyrimidines. If so desired, two or more universalbases can be included at a particular position in a probe. A number ofuniversal bases are known in the art including, but not limited to,hypoxanthine, 3-nitropyrrole, 4-nitroindole, 5-nitroindole,4-nitrobenzimidazole, 5-nitroindazole, 8-aza-7-deazaadenine,6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P. Kong Thoo Lin. andD. M. Brown, Nucleic Acids Res., 1989, 17, 10373-10383),2-amino-6-methoxyaminopurine (D. M. Brown and P. Kong Thoo Lin,Carbohydrate Research, 1991, 216, 129-139), etc. Hypoxanthine is onepreferred fully universal base. Nucleosides comprising hypoxanthineinclude, but are not limited to, inosine, isoinosine, 2′-deoxyinosine,and 7-deaza-2′-deoxyinosine, 2-aza-2′deoxyinosine. Naturally occurringand synthetic analogs may also be used, including for examplehypoxanthine, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-N⁴ethencytosine, 4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy[3,4-d]pyrimidine, among others. The nucleotide unitsof the oligonucleotides may also have a cross-linking function (e.g. analkylating agent).

A nucleoside is usually a compound having a nucleotide base covalentlylinked to the C-1′ carbon of a pentose sugar. In certain embodiments,the linkage is via a heteroaromatic ring nitrogen. Typical pentosesugars include, but are not limited to, those pentoses in which one ormore of the carbon atoms are each independently substituted with one ormore of the same or different —R, —OR, —NRR or halogen groups, whereeach R is independently hydrogen, (C₁-C₆) alkyl or (C₅-C₁₄) aryl. Thepentose sugar may be saturated or unsaturated. Exemplary pentose sugarsand analogs thereof include, but are not limited to, ribose,2′-deoxyribose, 2′-(C₁-C₆)alkoxyribose, 2′-(C₅-C₁₄)aryloxyribose,2′,3′-dideoxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C₁-C₆)alkylribose,2′-deoxy-3′-(C₁-C₆)alkoxyribose and 2′-deoxy-3′-(C₅-C₁₄)aryloxyribose.One or more of the pentose carbons of a nucleoside may be substitutedwith a phosphate ester, as disclosed in, for example, U.S. Pat. No.7,255,994. In certain embodiments, the nucleosides are those in whichthe nucleotide base is a purine, a 7-deazapurine, a pyrimidine, auniversal nucleotide base, a specific nucleotide base, or an analogthereof. Nucleotide analogs include derivatives in which the pentosesugar and/or the nucleotide base and/or one or more of the phosphateesters of a nucleoside may be replaced with its respective analog.Exemplary pentose sugar analogs and nucleotide base analog are describedabove. Exemplary phosphate ester analogs include, but are not limitedto, alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, etc., and mayinclude associated counterions. Other nucleotide analogs are nucleotideanalog monomers which can be polymerized into polynucleotide analogs inwhich the DNA/RNA phosphate ester and/or sugar phosphate ester backboneis replaced with a different type of linkage. Exemplary polynucleotideanalogs include, but are not limited to, peptide nucleic acids, in whichthe sugar phosphate backbone of the polynucleotide is replaced by apeptide backbone. The internucleoside linkages can be a phosphodiesterlinkage, although other linkages (e.g., scissile linkages which can besubstantially cleaved under conditions in which phosphodiester linkagesare not substantially cleaved) can be used. For example, a linkage thatcontains an AP endonuclease sensitive site, for example an abasicresidue, a residue containing a damaged base that is a substrate forremoval by a DNA glycosylase, or another residue or linkage that is asubstrate for cleavage by an AP endonuclease, or a disaccharidenucleoside.

As used herein, the term “oligonucleotide” (“oligo”) or “polynucleotide”may refer to an oligomer of nucleotides or derivatives thereof.Polynucleotides include double- and single-stranded DNA, as well asdouble- and single-stranded RNA, DNA:RNA hybrids, peptide-nucleic acids(PNAs) and hybrids between PNAs and DNA or RNA, and also include knowntypes of modifications, for example, labels which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbonates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide. The oligomers may also includemodified bases, and/or backbones (e.g., modified phosphate linkage ormodified sugar moiety). Non-limiting examples of synthetic backbonesthat confer stability and/or other advantages to the oligomers mayinclude phosphorothioate linkages, peptide nucleic acid, locked nucleicacid (Singh, et al. Chem Commum 4:455-456 (1998)), xylose nucleic acid,and/or analogues thereof. In other cases, the polynucleotide can containnon-nucleotidic backbones, for example, polyamide (e.g., peptide nucleicacids (PNAs)) and polymorpholino (commercially available from theAnti-Virals, Inc., Corvallis, Oreg., as Neugene™ polymers), and othersynthetic sequence-specific nucleic acid polymers providing that thepolymers contain nucleobases in a configuration which allows for basepairing and base stacking, such as is found in DNA and RNA.

Oligonucleotides and/or polynucleotides may be any length “n.” Forexample, n may be any of 1, 2, 4, 6, 8, 12, 16, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40 etc. number of nucleotides. The polynucleotidestructure (N)_(n) represents an oligonucleotide consisting of n numberof nucleotides N (e.g., (I)₈ is representative of an oligonucleotidehaving the sequence IIIIIIII; or (A)₁₂ is representative of anoligonucleotide having the sequence AAAAAAAAAAAA). Other types ofoligonucleotides or polynucleotides may also be suitable for use aswould be understood to one of skill in the art from this disclosure.

Oligonucleotides and/or polynucleotides may optionally be attached toone or more non-nucleotide moieties such as labels and other smallmolecules, large molecules such proteins, lipids, sugars, and solid orsemi-solid supports, for example through either the 5′ or 3′ end. Labelsinclude any moiety that is detectable using a detection method ofchoice, and thus renders the attached nucleotide or polynucleotidesimilarly detectable using a detection method of choice (e.g., using aSGC and/or detectable label). Optionally, the label emitselectromagnetic radiation that is optically detectable or visible. Insome cases, the nucleotide or polynucleotide is not attached to a label,and the presence of the nucleotide or polynucleotide is directlydetected.

As used herein, the term “nucleic acid” refers to polymers ofnucleotides or derivatives thereof. As used herein, the term “targetnucleic acid” refers to a nucleic acid that is desired to be amplifiedin a nucleic acid amplification reaction. For example, the targetnucleic acid comprises a nucleic acid template. In some embodiments, thetarget nucleic acid may be the product of the ligation of at least twooligonucleotides to one another.

As used herein, the term “sequence” refers to a nucleotide sequence ofan oligonucleotide or a nucleic acid. Throughout the specification,whenever an oligonucleotide/nucleic acid is represented by a sequence ofletters, the nucleotides are in 5′ to 3′ order from left to right. Forexample, if the polynucleotide contains bases Adenine, Guanine,Cytosine, Thymine, or Uracil, the polynucleotide sequence can berepresented by a corresponding succession of letters A, G, C, T, or U),e.g., a DNA or RNA molecule. And, an oligonucleotide represented by asequence (I)_(n)(A)_(n) wherein n=1, 2, 3, 4 and so on, represents anoligonucleotide where the 5′ terminal nucleotide(s) is inosine and the3′ terminal nucleotide(s) is adenosine.

Oligonucleotides and/or polynucleotides can optionally be regarded ashaving “complementary” sequences if the same may hybridize to oneanother. The term “hybridization” typically refers to the process bywhich oligonucleotides and/or polynucleotides become hybridized to eachother. The adjectival term “hybridized” refers to two polynucleotideswhich are bonded to each other by two or more sequentially adjacent basepairings. Typically, these terms refer to “specific hybridization”. Twooligonucleotides and/or polynucleotides may selectively (orspecifically) hybridize to each other if they bind significantly ordetectably to each other under stringent hybridization conditions whenpresent in a complex polynucleotide mixture such as total cellular orlibrary DNA. In some embodiments, for selective or specifichybridization, a positive signal is at least two times background,preferably 10 times background hybridization. Optionally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point for the specific sequence at a defined ionic strength pH.Stringent conditions are optionally in which the salt concentration isless than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodiumion concentration (or other salts) at pH 7.0 to 8.3 and the temperatureis at least about 30° C. for short probes (e.g., 10 to 50 nucleotides)and at least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides which they encode aresubstantially identical. “Nonspecific hybridization” is used to refer toany unintended or insignificant hybridization, for example hybridizationto an unintended polynucleotide sequence other than the intended targetpolynucleotide sequence. The uninentended polynucleotide sequence can beon the same or different polynucleotide from the intended target. Insome cases, the only intended hybridization can be from Watson-Crickbase pairing between two polynucleotides. Other kinds of intended basepairings can include base pairing between corresponding analogs of suchnucleotides or between iso-cytidine and iso-guanine. In some cases wherehybridization is only intended between complementary bases, any bondingbetween non-complementary bases is considered to be non-specifichybridization.

In some embodiments, complementary sequences may be those that, whenhybridized together, may be efficiently ligated to a thirdpolynucleotide that has hybridized adjacently to it. Similarly,nucleotide residues can be regarded as complementary if when both arebase-paired with each other within two hybridized polynucleotides,either nucleotide can be ligated in a template-driven ligation reactionwhen situated as the terminal nucleotide in its polynucleotide.Nucleotides that are efficiently incorporated by DNA polymerasesopposite each other during DNA replication under physiologicalconditions are also considered complementary. In an embodiment,complementary nucleotides can form base pairs with each other, such asthe A-T/U and G-C base pairs formed through specific Watson-Crick typehydrogen bonding between the nucleobases of nucleotides and/orpolynucleotides positions antiparallel to each other. Thecomplementarity of other artificial base pairs can be based on othertypes of hydrogen bonding and/or hydrophobicity of bases and/or shapecomplementarity between bases. In appropriate instances, polynucleotidescan be regarded as complementary when the same may undergo cumulativebase pairing at two or more individual corresponding positions inantiparallel orientation, as in a hybridized duplex. Optionally therecan be “complete” or “total” complementarity between a first and secondpolynucleotide sequence where each nucleotide in the firstpolynucleotide sequence can undergo a stabilizing base pairinginteraction with a nucleotide in the corresponding antiparallel positionon the second polynucleotide. “Partial” complementarity describespolynucleotide sequences in which at least 20%, but less than 100%, ofthe residues of one polynucleotide are complementary to residues in theother polynucleotide. A “mismatch” is present at any position in the twoopposed nucleotides that are not complementary. In some ligation assays,a polynucleotide can undergo substantial template-dependent ligationeven when it has one or more mismatches to its hybridized template.Optionally, the polynucleotide has no more than 4, 3, or 2 mismatches,e.g., 0 or 1 mismatch, with its template. In some assays, thepolynucleotide will not undergo substantial template-dependent ligationunless it is at least 60% complementary, e.g., at least about 70%, 80%,85%, 90%, 95% or 100% complementary to its template.

“Degenerate”, with respect to a position in a polynucleotide that is oneof a population of polynucleotides, means that the identity of the baseof the nucleoside occupying that position varies among different membersof the population. A population of polynucleotides in this context isoptionally a mixture of polynucleotides within a single continuous phase(e.g., a fluid). The “position” can be designated by a numerical valueassigned to one or more nucleotides in a polynucleotide, generally withrespect to the 5′ or 3′ end. For example, the terminal nucleotide at the3′ end of an extension probe may be assigned position 1. Thus in a poolof extension probes of structure 3′-XXXNXXXX-5′, the N is at position 4.A position is said to be k-fold degenerate if it can be occupied bynucleosides having any of k different identities. For example, aposition that can be occupied by nucleosides comprising either of 2different bases is 2-fold degenerate.

A “solid support”, as used herein, typically refers to a structure ormatrix on or in which ligation and/or amplification reagents (e.g.,nucleic acid molecules, microparticles, and/or the like) may beimmobilized so that they are significantly or entirely prevented fromdiffusing freely or moving with respect to one another. The reagents canfor example be placed in contact with the support, and optionallycovalently or noncovalently attached or partially/completely embedded.The terms “microparticle,” “beads”, “microbeads”, etc., refer toparticles (optionally but not necessarily spherical in shape) having asmallest cross-sectional length (e.g., diameter) of 50 microns or less,preferably 10 microns or less, 3 microns or less, approximately 1 micronor less, approximately 0.5 microns or less, e.g., approximately 0.1,0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer, about1-10 nanometer, about 10-100 nanometers, or about 100-500 nanometers).Microparticles (e.g., Dynabeads from from Dynal, Oslo, Norway) may bemade of a variety of inorganic or organic materials including, but notlimited to, glass (e.g., controlled pore glass), silica, zirconia,cross-linked polystyrene, polyacrylate, polymehtymethacrylate, titaniumdioxide, latex, polystyrene, etc. Magnetization can fecilitatecollection and concentration of the microparticle-attached reagents(e.g., polynucleotides or ligases) after amplification, and facilitatesadditional steps (e.g., washes, reagent removal, etc.). In certainembodiments of the invention a population of microparticles havingdifferent shapes sizes and/or colors can be used. The microparticles canoptionally be encoded, e.g., with quantum dots such that eachmicroparticle can be individually or uniquely identified.

As used herein the term “reaction mixture” refers to the combination ofreagents or reagent solutions, which are used to carry out a chemicalanalysis or a biological assay. In some embodiments, the reactionmixture comprises all necessary components to carry out a nucleic acid(DNA) synthesis/amplification reaction. As described above, suchreaction mixtures may include at least one amplification primer pairsuitable for amplifying a nucleic acid sequence of interest (e.g.,target nucleic acid). As described above, a suitable reaction mixturemay also include a “master mix” containing the components (e.g.,typically not including the primer pair) needed to perform anamplification reaction (e.g., detergent, magnesium, buffer components,etc.). Other embodiments of reaction mixtures are also contemplatedherein as would be understood by one of skill in the art.

As used herein, the terms “reagent solution” or “solution suitable forperforming a DNA synthesis reaction” refer to any or all solutions,which are typically used to perform an amplification reaction or DNAsynthesis. They include, but are not limited to, solutions used in DNAamplification methods, solutions used in PCR amplification reactions, orthe like. The solution suitable for DNA synthesis reaction may comprisebuffer, salts, and/or nucleotides. It may further comprise primersand/or DNA templates to be amplified. One or more reagent solutions aretypically included in the reactions mixtures or master mixes describedherein.

As used herein, the term “primer” or “primer sequence” refers to a shortlinear oligonucleotide that hybridizes to a target nucleic acid sequence(e.g., a DNA template to be amplified) to prime a nucleic acid synthesisreaction. The primer may be a RNA oligonucleotide, a DNAoligonucleotide, or a chimeric sequence (e.g., comprising RNA and DNA).The primer may contain natural, synthetic, or modified nucleotides. Boththe upper and lower limits of the length of the primer are empiricallydetermined. The lower limit on primer length is the minimum length thatis required to form a stable duplex upon hybridization with the targetnucleic acid under nucleic acid amplification reaction conditions. Veryshort primers (usually less than 3 nucleotides long) do not formthermodynamically stable duplexes with target nucleic acid under suchhybridization conditions. The upper limit is often determined by thepossibility of having a duplex formation in a region other than thepre-determined nucleic acid sequence in the target nucleic acid.Generally, suitable primer lengths are in the range of about any of, forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, (and so on) nucleotides in length.

In some embodiments, the terms “probe(s)”, “oligonucleotide(s)” and/or“primer(s)” may be interchangeable terms herein, so that any one ofthese may be taken as a reference to another. The terms“polynucleotide”, “oligonucleotide”, “probe”, “primer”, “template”,“nucleic acid” and the like may be taken to refer to a populations orpools of individual molecules that are substantially identical acrosstheir entire length or across a relevant portion of interest. Forexample, the term “template” may indicate a plurality of templatemolecules that are substantially identical, etc. In the case ofpolynucleotides that are degenerate at one or more positions, it will beappreciated that the degenerate polynucleotide may comprise a pluralityof polynucleotide molecules, which have sequences that are substantiallyidentical only at the nondegenerate position(s) and differ in sequenceat the degenerate positions. Thus, reference to “a” polynucleotide(e.g., “a” primer, probe, oligonucleotide, template, etc.) may be takento mean a population of substantially identical polynucleotidemolecules, such that the plural nature of a population of substantiallyidentical nucleic acid molecules need not be explicitly indicated, butmay if so desired. These terms are also intended to provide adequatesupport for a claim that explicitly specifies a single polynucleotidemolecule itself.

“Ligation” involves the formation of a covalent bond or linkage betweenthe termini of two or more nucleic acids, e.g. oligonucleotides and/orpolynucleotides, optionally in a template-driven reaction. Exemplaryligations may be carried out enzymatically to form a phosphodiesterlinkage between a 5′ carbon of a terminal nucleotide of oneoligonucleotide with 3′ carbon of another oligonucleotide (e.g., using aligase). The nature of the bond or linkage may vary widely and theligation is preferably achieved enzymatically. The efficiency ofligation refers to the rate of ligation. Where the relative efficiencyof ligation is specified in comparative or relative terms by comparisonto a reference ligation assay, it is implicit that all other reagentsand conditions (e.g., temperature, concentration of all reagents, pH,concentration of requisite ions such as Mg++ and Mn++, concentration ofrequisite cofactors such as NAD and/or ATP, salts, buffers, molarconcentrations of all reagents, including enzyme, template, probeprimer, oligonucleotides, etc) are otherwise kept identical. Forexample, a proviso that a ligase (e.g., an SFL (see below)) can ligate ashort (e.g., less than 6 nucleotides) probe at least X % (e.g., where Xis 100 or less; 100, 99, 95, 90, 85, 80, 75, 70, 50, 25, 10 1, 0.5, 0.1,etc., or any increment in between) as efficiently as the ligase canligate a corresponding octanucleotide, may be understood to mean thatthe rate of ligation of the shorter probe occurs at a rate that is atleast X % of the rate of ligation of the octanucleotide, where allreagents except for the probes (e.g., primer, template, enzymes and anyother reagents) and all reaction conditions (e.g., temperature, reagentconcentrations, concentrations of any other reagents, etc) are keptinvariant for practical purposes. It is understood that ligationefficiency in absolute or relative terms may increase or decreasedepending on the exact reaction conditions used.

Optionally, ligation is performed under in-vitro conditions that havebeen experimentally determined to be suitable or optimal for ligaseactivity. Preferably, reaction conditions are kept substantially similarto in-vivo or physiological conditions in which a naturally-occurringform of the ligase being used is naturally active. Most preferably, thereaction conditions for a particular ligase are matched as closely aspossible to exemplary in vitro ligation conditions described herein forthat ligase. In other embodiments, the conditions are such that thereference ligation assay produces significant or detectable ligationwithin 30 minutes, within 10 minutes, within 1 minute, or within tenseconds. Another non-limiting example of a significant or detectablerate of ligation generates in the range of 100 pM of ligation product,optionally about 1000 pM or 10,000 pM, in an appropriate amount of time(e.g., 10 minutes).

Along similar lines, it should be understood that a statement that aresult has occurred (e.g., ligation, binding) is intended to indicatethat the result has occurred at a significant or substantial level or anenhanced level compared to when it has not occurred. For example,ligation is said to have not occurred if it is not significant,insubstantial or greatly reduced (e.g., reduced by at least 80%, 90%,95% or 99% compared to when ligation does occur (e.g., under theconditions described in the last paragraph). In reference to ligation oftwo polynucleotides, the “proximal” terminus of either polynucleotide isther terminus that is intended to be ligated to the otherpolynucleotide. It is generally the terminus that is closer to the otherpolynucleotide, or the terminus that is contacted by the active site ofthe ligase, or ther terminus that is eventually ligated to the otherpolynucleotide, while the opposite terminus is the “distal” terminus.The terminal nucleotide residue at the proximal terminus can be termedthe proximal nucleotide, and the proximal nucleotide position optionallydesignated as position 1, the penultimate nucleotide position asposition 2, etc. In some non-limiting instances of template-dependentligation, the proximal termini of both polynucleotides are hybridizedadjacently to each other.

An exemplary type of enzymatic ligation (double-stranded ligation)includes the formation of a covalent bond between nucleotides of apolynucleotide (e.g., resulting in circularization) or between two ormore polynucleotides (e.g., a first double-stranded terminus of a firstpolynucleotide and a second different double-stranded terminus of asecond polynucleotide). The polynucleotides may be different, or may bethe same. Polynucleotides may also be ligated using a “splint”oligonucleotide which may be used to link nucleotides that the userdesires to ligate (e.g., on the same or different polynucleotides).Optionally, the ends of both double-stranded termini may be joinedirrespective of their sequences (e.g., blunt-end ligation, ornon-homologous end joining).

In another variation, two double-stranded polynucleotides withprotruding single-stranded ends that are complementary to each other canbe ligated (e.g., cohesive-end ligation). In other instances, theligation can ligate two single-stranded polynucleotides, either or bothof which has optionally hybridized (annealed) to another nucleotidesequence. In template-dependent ligation, ligation between a firstpolynucleotide and a second polynucleotide occurs upon hybridization ofat least a portion of either or both polynucleotides to a targetsequence. The target sequence can be a portion of either polynucleotide(e.g., self-hybridization or hybridization to each other) or to asequence on a third different polynucleotide (e.g., a “splint”oligonucleotide). The hybridized portion of the polynucleotide may be,for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20nucleotides long. The hybridized portion is optionally a terminalportion of the nucleotide (e.g., includes the 5′ or 3′ nucleotide). Forexample, the hybridized portion can consist of the 5′ or 3′ terminalnucleotide, or the terminal 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20nucleotides of the 5′ or 3′ end. Optionally, ligation occurs when nomismatch is present within the hybridized portions.

In other cases, ligation occurs when one, two or three mismatches can bepresent within the hybridized portion. In some cases ligation does notoccur when the terminal nucleotide and/or second-most terminalnucleotide and/or third-most terminal nucleotide is mismatched. Asmentioned, the terminal nucleotides can be the 5′- or 3′-terminalnucleotides of the polynucleotide. An exemplary type of assay makes useof template-dependent ligation between a first single-strandedpolynucleotide and a second single-stranded polynucleotide, whereligation can be effected when either or both polynucleotides is/arehybridized to a third different single-stranded polynucleotide. In someinstances, both probes must hybridize to the template for significantligation to occur. For ease of reference, the first polynucleotide iscalled the “initializing probe,” the second polynucleotide called the“extension probe” and the third polynucleotide called the “template.”

In some variations, (e.g., “nick ligation”), both probes must hybridizeadjacently to each other on the template for ligation to occur. In someassays, the probes are adjacently hybridized and can be ligated onlywhen a terminal nucleotide of the initializing probe is hybridized to afirst nucleotide of the template and a terminal nucleotide of theextension probe is hybridized to a second nucleotide of the template,where the first and second nucleotides on the template are not separatedby an intervening nucleotide of the template. In other embodiments, afew intervening nucleotides may be present between the first and secondnucleotides on the template (e.g., 1, 2, 3 or more nucleotides). In suchembodiments, a “gap-filling” step can be performed to extend the 3′terminus of one probe before it can be ligated to the 5′ terminus of theother probe.

In the methods described herein, the terminal nucleotide of theinitializing probe can be the 5′ terminal nucleotide and the terminalnucleotide of the extension probe can be the 3′ terminal nucleotide.Alternatively, the terminal nucleotide of the initializing probe can bethe 3′ terminal nucleotide and the terminal nucleotide of the extensionprobe can be the 5′ terminal nucleotide. The ligation product of any onereaction can optionally be subjected to further ligation and/ornon-ligation reactions in turn. For example, the ligation product can beused as the initializing probe or extension probe or template in asubsequent ligation. Also for example, it can be used as a template orprimer for polymerase extension, such as in polymerase chain reaction(PCR). It can be cleaved enzymatically or chemically (for example whenit has scissile linkages), treated with exo- or endonucleases, kinases,phosphatases, etc. The ends of a double-stranded product can beblunt-ended or filled in, capped, or adenylated, etc.

As used herein, “splint oligonucleotide,” “splint oligo,” or “connector”refers to an oligonucleotide that is used to provide an annealing siteor a “ligation template” for joining two ends of a nucleic acid moleculeor molecules using a ligase or another enzyme with ligase activity. Theligation splint holds the ends adjacent to each other and “creates aligation junction” between the 5′-phosphorylated and a 3′-hydroxylatedends that are to be ligated. For example, when a ligation splint oligois used to join the 3′-end of a first probe oligo (oligo A) to the5′-end of a second probe oligo, the ligation splint oligo has a sequencecomplementary to the 3′-end of oligo A (e.g., oligo tail sequence, and asecond neighboring sequence (e.g., an adjacent sequence) that iscomplementary to the 5′-end of oligo B (FIG. 4A and FIG. 4B).

In some embodiments of the improved PLA processes splint oligos can beeither symmetrical or asymmetrical depending on the number ofnucleotides that hybridize to each of the two oligo probes it isconnecting or ligating. FIG. 4A and FIG. 4B diagrams asymmetrical andsymmetrical splint types for use in the improved PLA processes asdescribed herein. In some embodiments, asymmetrical splints (or“connectors”) span across the two separate oligo probes (e.g., probeoligo A and B) with one of the ends of the splint (e.g., either the3′-end or the 5′-end) having more nucleotides that hybridize to one ofthe probe oligos than the other end of the splint has nucleotides thathybridize to the alternative probe oligo (FIG. 4A). In otherembodiments, symmetrical splints span across the two separate oligoprobes (e.g., probe oligo A and B) with both ends of the splint (e.g.,the 3′ end and the 5′ end) having equal number of nucleotides thathybridize to each of the two probe oligos (FIG. 4B).

Both asymmetrical and symmetrical splints can have any number ofintervening nucleotides between each of its 3′ and 5′ ends thathybridize to the separate probe oligos. Alternatively, there may be nointervening nucleotides between each of the 3′ and 5′ ends thathybridize to the probe oligos. In preferred embodiments, splintoligonucleotides (oligos) are at least 6 nucleotides long (e.g., 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In certainother embodiments, each of the 3′- or 5′-ends of the splint oligo willcomprise at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotidesthat separately hybridize to (or “overlap”) an oligo probe, hereinreferred to as the “overhang” region.

In some embodiments of the improved PLA processes the splint oligo isblocked at its 3′-end. The blocking agent can be any covalentlyconnected moiety that prevents polymerase activity. This 3′ blockedsplint oligo is then prevented from interfering with the PCR reactionpart of the improved PLA process. In some embodiments, for example, the3′ blocking agent can include, but is not limited to, 3′-fluoro-,3′-bromo-, 3′-iodo-, 3′-deoxy-, 3′-methyl-, 3′-methoxy, 3′-phosphate,3′-aminolink, 3′-abasic amidite, or any other 3′ modification groups.Those of ordinary skill in the art would be able to further contemplateother blocking agents for use as disclosed herein.

Any one or more of the ligation methods provided herein can be used in aligation assay. Non-limiting example of ligation assays include aoligonucleotide ligation assay (OLA), a ligase chain reaction (LCR), aligase detection reaction (LDR) and combination assays such as the OLAcoupled with the polymerase chain reaction (PCR), e.g., OLA-PCR andPCR-OLA, the Combined Chain Reaction (CCR; a combination of PCR and LCR)and PCR-LDR (see, e.g., Landegren et al., Science 241:1077-80, 1988;Barany, Proc. Natl. Acad. Sci. 88:189-93, 1991; Grossman et al., Nucl.Acids Res. 22(21):4527-34, 1994; Bi and Stambrook, Nucl. Acids Res.25(14):2949-51, 1997; Zirvi et al., Nucl. Acids Res., 27(24):e40, 1999;U.S. Pat. No. 4,988,617; and PCT Publication Nos. WO 97/31256 and WO01/92579. Such assays have been used for single nucleotide polymorphism(SNP) analysis, SNP genotyping, mutation detection, identification ofsingle copy genes, detecting microsatellite repeat sequences, and DNAadduct mapping, among other things. See also Whitely et al, U.S. Pat.No. 4,883,750; Letsinger et al, U.S. Pat. No. 5,476,930; Fung et al,U.S. Pat. No. 5,593,826; Kool, U.S. Pat. No. 5,426,180; Landegren et al,U.S. Pat. No. 5,871,921; Xu and Kool, Nucleic Acids Research, 27:875-881 (1999); Higgins et al, Methods in Enzymology, 68: 50-71 (1979);Engler et al, The Enzymes, 15-3-29 (1982); and Namsaraev, U.S. Pat. Pub.2004/0110213. The fidelity of several known ligases, based on forexample the evaluation of mismatch ligation or ligation rates, has beenreported. For example, the NAD+-dependent ligase from thehyperthermophilic bacteria Aquifex aeolicus reportedly generatesdetectable 3′ misligation products with C:A, T:G, and G:T mismatches(Tong et al., Nucl. Acids Res. 28(6):1447-54, 2000); a partiallypurified preparation of bovine DNA ligase III reportedly generateddetectable 3′ misligation products with C:T, G:T, and T:G mismatches,while human ligase I generated detectable 3′ misligation products withC:T and G:T mismatches, but not T:G mismatches (Husain et al., J. Biol.Chem. 270(16):9683-90, 1995); and the DNA ligase from the thermophilicbacteria Thermus thermophilus (Tth) reportedly generates detectablelevels of 3′ misligation products with T:G and G:T mismatches (Luo etal., Nucl. Acids Res. 24(14):3071-78, 1996). Bacteriophage T4 DNA ligasereportedly generates detectable misligation products with a wide rangeof mismatched substrates and appears to have lower fidelity than Thermusspecies ligases by at least one to two orders of magnitude (Landegren etal., Science 241:1077-80, 1988; Tong et al., Nucl. Acids Res.27(3):788-94, 1999).

A particularly useful assay is the oligonucleotide ligation assay (OLA).The OLA is a convenient, highly-stringent method that permitsdistinction among known DNA sequence variants (Landegren, 1988). Forinstance, multiplex analysis of highly polymorphic loci is useful foridentification of individuals, e.g., for paternity testing and inforensic science, organ transplant donor-receiver matching, geneticdisease diagnosis, prognosis, and pre-natal counseling, and othergenetic-based testing which depend on the discrimination of single-basedifferences at a multiplicity of loci (Delahunty, 1996). Products of amultiplex OLA may be resolved electrophoretically from one another andfrom unligated probes under denaturing conditions with fluorescencedetection (Grossman, 1994). For example, two PNA-DNA chimeras, awild-type (WT) sequence chimera and a mutant sequence chimera, may beardifferent fluorescent dyes. Only when the mutant sequence is present inthe target sample, will the mutant sequence chimera ligate to theadjacently annealed second probe (oligo) if the mutant base pair is atthe ligation site. The ligation products may be discriminated byseparation based on: (i) size using electrophoresis or chromatographyand/or (ii) detectable labels (Grossman, 1994). With a plurality offluorescent dyes labeled to chimeras with sequences targeting uniquetarget sequences, multiplexed OLA can be conducted on a single sample ina single vessel. Requirements for efficient multiplex OLA include probesthat anneal and ligate in a highly specific and rapid manner. Thechimeras and second probe sequences may be selected such that the mutantbase, or single base polymorphism, may be at the 5′-phosphate of thesecond probe or the 3′-terminus of the chimera. It is contemplated thatOLA experiments of the present invention may be conducted on solidsupports where the template nucleic acid, PNA-DNA chimeric probe, or thesecond probe may be immobilized on a solid particle or bead, or a solidporous or non-porous surface. When immobilized, the template, chimera orsecond probe is preferably covalently attached to the solid substrate,e.g. via a terminal monomer unit. The solid substrate may bepolystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide,magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide,polyethylene, polyethyleneoxy, and copolymers and grafts of any of theabove solid substrates. The configuration or format of the solidsubstrate may be small particles or beads of approximately 1 to 50 μm indiameter, membranes, frits, slides, plates, micromachined chips,alkanethiol-gold layers, non-porous surfaces, andpolynucleotide-immobilizing media.

As described above, enzymatic ligation is typically accomplished using aligase, which may be a polypeptide. Suitable ligases include, forexample, nucleic acid ligase, oligonucleotide ligase, DNA ligase, RNAligase, and the like. Suitable RNA ligases include those described orused in, for example, any of U.S. Pat. Nos. 4,582,802; 5,665,545;6,194,637; 6,444,429; 6,455,274; 6,576,453; 6,635,425; 6,855,523;7,811,753; and the like, and/or any of U.S. Pat. Pubs. 2004/0171047A1,2004/0191871A1, 2005/0266487A1, 2006/0223098A1, 2007/0037190A1,2008/0160526A1; 2009/0061481A1, 2010/0099683A1, and/or 2010/0184618A1;and the like, all of which are incorporated by reference in theirentirety into this application. Exemplary DNA ligases may include, forexample, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7 DNA ligase,vaccinia virus DNA ligase, E. coli DNA ligase, mammalian DNA ligase I,mammalian DNA ligase II, mammalian DNA ligase III, Tth DNA ligase, KODDNA ligase, a thermostable DNA ligase, and/or derivatives, fragments,and/or combinations thereof. Suitable RNA ligases include thosedescribed or used in, for example, any of U.S. Pat. Nos. 4,661,450;5,516,664; 5,602,000; 5,807,674; 6,368,801; 6,492,161; 6,635,453; andthe like, or any of U.S. Pat. Pub. Nos. 2003/0082536A1; 2004/0058330A1;2005/0266439A1; 2005/0074774A1; 2008/0045418A1; 2010/00159526A1; and thelike, all of which are incorporated by reference in their entirety intothis application. Exemplary RNA ligases may include, for example, T4 RNAligase, bacteriophage RB69 RNA ligase, Autographa californicanuclearpolyhedrosis virus RNA ligase, a thermophilic RNA ligase,bacteriophage RM378 RNA ligase, bacteriophage TS2126 RNA ligase, and/orderivatives, fragments, and/or combinations thereof.

In some embodiments, the ligase is a “small footprint ligase” (SFL). ASFL has the the ability to ligate short polynucleotides (e.g., at leastabout 3 nucleotides). As described herein, a SFL may ligateoligonucleotides having a connector oligo length of as short as 3 baseof hybridized DNA adjacent to 5′-phosphate hybridized DNA. In someembodiments, the SFL may be used to ligate oligonucleotides comprisingshort overlap sequences (e.g., short connector oligo length). Forinstance, the SFL may be used to ligate oligonucleotides of variousnucleotides in length, whereby each oligo has at least a 3 nucleotideoverlap with the splint oligo. Typical ligases would be better suitedfor ligating longer oligonucleotides (e.g., comprising 9 or morenucleotides; comprising 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, etcnucleotides). In this way, oligonucleotide concentration may also bereduced to minimize chance of solution hybridization promotednon-antigen-binding ligation. For combining the ligation and PCRreaction into one step, ATP (cofactor for the ligase) can be omittedfrom the reaction mixture. In order to maintain the ligase function, theSFL may be pre-enriched with ATP prior to its purification and use.

A SFL may be a naturally occurring or non-naturally occurring (e.g.,artificial, synthetic) ligase. A SFL can comprise a polypeptide sequencethat is homologous to or a variant of a known ligase sequence or anyportion thereof. Exemplary SFLs can have amino acid sequence identity ofat least 70%, optionally at least 85%, optionally at least 90 or 95%,with a known ligase, and possesses one or more functional activities ofa ligase. A SFL can thus comprise a polypeptide having any one or moreof the following activities: (1) nucleophilic attack on ATP or NAD⁺resulting in release of PPi or NMN and formation of a covalentligase-adenylate intermediate; (2) transferring the adenylate to the5′-end of the 5′-phosphate-terminated DNA strand to form DNA-adenylate(e.g., the 5′-phosphate oxygen of the DNA strand attacks the phosphorusof ligase-adenylate); and, (3) formation of a covalent bond joining thepolynucleotide termini and liberation of AMP (e.g., by the attack by the3′-OH on DNA-adenylate). Optionally, the SFL can mediate any one or moreof the following bond transformations: from phosphoanhydride (ATP) tophosphoramidate (ligase-adenylate); from phosphoramidate(ligase-adenylate) to phosphoanhydride (DNA-adenylate); and/or fromphosphoanhydride (DNA-adenylate) to phosphodiester (sealed DNA). The SFLin one aspect is an enzyme that can mediate the formation of a covalentbond between two polynucleotide termini, e.g., a 3′-OH terminus and a5′-PO₄ terminus are joined together to form a phosphodiester bond. Insome instances, DNA ligation entails any one or more of three sequentialnucleotidyl transfer steps, discussed below. All three chemical stepsdepend on a divalent cation cofactor. In one aspect, the SFL is anATP-dependent ligase or a NAD⁺-dependent ligase.

For example, the SFL can comprise any one or more domains characteristicof a ligase (e.g., an N-terminal nucleotidyltransferase (NTase) domainand/or a C-terminal OB domain). The OB domain optionally comprises afive-stranded antiparallel beta-barrel plus an alpha-helix. Within theNTase domain is an adenylate-binding pocket composed of the six peptidemotifs that define the covalent NTase enzyme family of polynucleotideligases. Optionally, the NTase domain can comprise any one or more ofthe ligase amino acid motifs I, III, IIIa, IV, and/or V, and preferablyall six motifs. Motif I (e.g., KxDGxR or a “KXDG” motif) optionallycontains a lysine. Exemplary sequences for each motif in CV ligase areATPKIDGIR (motif I) (SEQ ID NO.: 1), SRT (motif Ia), EGSDGEIS (motifIII) (SEQ ID NO.: 2), YWFDY (motif IIIa) (SEQ ID NO.: 3), EGVMIR (motifIV) (SEQ ID NO.: 4), LLKMK (motif V) (SEQ ID NO.:5). Motif 1 pfycontains a lysine residue. Other examples of motif I include CELKLDGLA(SEQ ID NO.: 6), VEHKVDGLS (SEQ ID NO.: 7), CEPKLDGLA (SEQ ID NO.: 8),CELKLDGVA (SEQ ID NO.: 9), AEIKYDGVR (SEQ ID NO.: 10), CEYKYDGQR (SEQ IDNO.: 11), VDYKYDGER (SEQ ID NO.: 12), FEIKYDGAR (SEQ ID NO.: 13),FEGKWDGYR (SEQ ID NO.: 14), AREKIHGTN (SEQ ID NO.: 15), ACEKVHGTN (SEQID NO.: 16), ILTKEDGSL (SEQ ID NO.: 17), and VEEKVDGYN (SEQ ID NO.: 18).Examples of motif Ia include TRG, SRT, SRR, SRN, SRS, KRT, KRS, SKG andTRG. Examples of motif III include LEVRGEVF (SEQ ID NO.: 19), VEVRGECY(SEQ ID NO.: 20), LEVRGEVY (SEQ ID NO.: 21), LEARGEAF (SEQ ID NO.: 22),FMLDGELM (SEQ ID NO.: 23), EGSDGEIS (SEQ ID NO.: 24), FILDTEAV (SEQ IDNO.: 25), FIIEGEIV (SEQ ID NO.: 26), AIVEGELV (SEQ ID NO.: 27), VVLDGEAV(SEQ ID NO.: 28), YQVFGEFA (SEQ ID NO.: 29), LVLNGELF (SEQ ID NO.: 30),FTANFEFV (SEQ ID NO.: 31) and LILVGEMA (SEQ ID NO.: 32). Examples ofmotif IIIa include FCYGV (SEQ ID NO.: 33), FLYTV (SEQ ID NO.: 34), TFYAL(SEQ ID NO.: 35), ICHGL (SEQ ID NO.: 36), NAYGI (SEQ ID NO.: 37), FVYGL(SEQ ID NO.: 38), KLYAI (SEQ ID NO.: 39), YWFDY (SEQ ID NO.: 40), YAFDI(SEQ ID NO.: 41), FLFDL (SEQ ID NO.: 42), NLFDV (SEQ ID NO.: 43), WAFDL(SEQ ID NO.: 44), YVFDI (SEQ ID NO.: 45), FAFDI (SEQ ID NO.: 46), ILLNA(SEQ ID NO.: 47), and FLFDV (SEQ ID NO.: 48). Examples of motif IVinclude DGVVIK (SEQ ID NO.: 49), DGIVIK (SEQ ID NO.: 50), DGVVVK (SEQ IDNO.: 51), DGTVLK (SEQ ID NO.: 52), EGLIVK (SEQ ID NO.: 53), EGVMIR (SEQID NO.: 54), EGLMVK (SEQ ID NO.: 55), EGVMVK (SEQ ID NO.: 56), EGLMAK(SEQ ID NO.: 57), EGVIAK (SEQ ID NO.: 58), EGYVLK (SEQ ID NO.: 59),EGVVIR (SEQ ID NO.: 60), EGYVAV (SEQ ID NO.: 61), and EGIIMK (SEQ IDNO.: 62). Examples of motif V include AVAFK (SEQ ID NO.: 63), AIAYK (SEQID NO.: 64), ALAYK (SEQ ID NO.: 65), AIAYK (SEQ ID NO.: 66), WWKMK (SEQID NO.: 67), LLKMK (SEQ ID NO.: 68), WLKLK (SEQ ID NO.: 69), WIKLK (SEQID NO.: 70), WLKIK (SEQ ID NO.: 71), WVKDK (SEQ ID NO.: 72), AIKCK (SEQID NO.: 73), IIKLR (SEQ ID NO.: 74), HFKIK (SEQ ID NO.: 75) and IVKYV(SEQ ID NO.: 76). The SFL optionally comprises all six motifs.Optionally all six motifs are found together in a naturally-occurringligase, such as a SFL identified herein. In some embodiments, the SFL isnot an RNA-capping enzyme. The ligase optionally comprises anyfunctional portion of a SFL. The ligase can be homologous to a SFL orany functional portion thereof, for example more than 75%, 85%, 90%, 95%or 99% homologous at the amino acid level. An exemplary SFL is aChlorella virus DNA ligase (ChVLig) (Ho, et al., J Virol,71(3):1931-19374 (1997)) or functional fragment or variant thereof.Representative examples of SFLs include CV ligase, DLX, DLXd, DLXd2 andMnM ligase. A preferred SFL is Chlorella Virus ligase. Some exemplaryligases are identified and their GI or accession numbers are provided inTABLE 1 below:

TABLE 1 PRK08224 B. Acidobacteria Bacteria; Fibrobacteres/Acidobacteriagroup; Acidobacteria; unclassifed Acidobacteria; Candidatus Koribacter;Candidatus Koribacter versatilis Candidatus Solibacter usitatusATP-Dependent YP_826317 Ellin6076Candidatus Solibacter (1 proteins) DNALigase C. Actinobacteria Bacteria; Actinobacteria; Actinobacteria(class); Actinobacteridae; Actinomycetales; Corynebacterineae;Mycobacteriaceae; Mycobacterium; Mycobacterium marinum Mycobacteriumgilvum PYR- ATP-Dependent YP_001132524 GCKMycobacterium (26 proteins)DNA Ligase Mycobacterium vanbaalenii PYR-1 ATP-Dependent YP_ 956315Mycobacterium (26 proteins) DNA Ligase Mycobacterium sp.MCSMycobacterium ATP-Dependent YP_642076 (26 proteins) DNA Ligase F.Chlamydiae/Verrucomicrobia Bacteria; Chlamydiae/Verrucomicrobia group;Verrucomicrobia; Opitutae; Opitutales; Opitutaceae; Opitutus; Opitutusterrae Opitutus terrae PB90-1Opitutus (1 proteins) ATP-DependentYP_001821013 DNA Ligase PRK09125 Organism Protein name Accession O.Betaproteobacteria Neisseria meningitidis Z2491Neisseria DNA ligaseYP_002341892 (7 proteins) Thiobacillus denitrificans ATCC DNA ligaseYP_314570 25259Thiobacillus (1 proteins) Variovorax paradoxusS110Variovorax DNA ligase YP_002944627 (1 proteins) Verminephrobactereiseniae DNA ligase YP_ 998235 EF01-2Verminephrobacter (1 proteins) P.Deltaproteobacteria Desulfobacterium autotrophicum LigA2 YP_002604477HRM2Desulfobacterium (1 proteins) Myxococcus xanthus DK 1622MyxococcusDNA ligase YP_628883 (1 proteins) Q. Epsilonproteobacteria Campylobacterjejuni sub sp. jejuni NCTC ATP-dependent YP_002345037 11168Campylobacter(10 proteins) DNA ligase Sulfurimonas denitrificans DSM DNA ligaseYP_393098 1251Sulfurimonas (1 proteins) R. GammaproteobacteriaAggregatibacter aphrophilus ATP-dependent YP_003007537NJ8700Aggregatibacter (2 proteins) DNA ligase Haemophilus influenzaePittEEHaemophilus ATP-dependent YP_001290961 (3 proteins) DNA ligaseShewanella baltica OS195Shewanella ATP-dependent YP_001554317 (18proteins) DNA ligase Shewanella loihica PV-4Shewanella ATP-dependentYP_001093713 (18 proteins) DNA ligase Vibrio cholerae M66-2Vibrio (9proteins) DNA ligase YP_002810248 PHA0454 Organism Protein nameAccession b. Viruses Viruses; dsDNA viruses, no RNA stage; Caudovirales;Podoviridae; Autographivirinae; phiKMV-like viruses Pseudomonas phageLKD16phiKMV-like viruses ATP-dependent YP_001522807 (7 proteins) DNAligase CLSZ2445448 Organism Protein name Accession a. EukaryotaEukaryota; Alveolata; Ciliophora; Intramacronucleata; Oligohymenophorea;Peniculida; Parameciidae; Paramecium; Paramecium tetraurelia Parameciumtetraurelia strain d4-2Paramecium DNA XP_001347270 (5 proteins) ligasePRK07636 Organism Protein name Accession J. Firmicutes Bacteria;Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus clausiiBacillus subtilis subsp. subtilis str. 168Bacillus ATP-dependentNP_389932 Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; DNAligase Geobacillus Geobacillus sp. Y412MC10Geobacillus ATP dependentYP_003240778 DNA ligase CLSK2551528 Organism Protein name Accession J.Firmicutes Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae;Geobacillus Geobacillus sp. Y412MC10Geobacillus (1 proteins) ATPdependent YP_003245332 DNA ligase CLSK2470953 Organism Protein nameAccession C. Actinobacteria Bacteria; Actinobacteria; Actinobacteria(class); Actinobacteridae; Actinomycetales; Micrococcineae;Micrococcaceae; Arthrobacter; Arthrobacter chlorophenolicus Arthrobacterchlorophenolicus A6 ATP dependent YP_002478427 (plasmid)Arthrobacter (2proteins) DNA ligase CLSK2469924 Organism Protein name Accession J.Firmicutes Bacteria; Firmicutes; Bacilli; Bacillales;Alicyclobacillaceae; Alicyclobacillus; Alicyclobacillus acidocaldarius;Alicyclobacillus acidocaldarius subsp. acidocaldarius Alicyclobacillusacidocaldarius subsp. acidocaldarius ATP dependent YP_003185050 DSM446Alicyclobacillus DNA ligase CLSK2340991 Organism Protein nameAccession N. Alphaproteobacteria Bacteria; Proteobacteria;Alphaproteobacteria; Caulobacterales; Caulobacteraceae;Phenylobacterium; Phenylobacterium zucineum Phenylobacterium zucineumHLK1 ATP-dependent YP_002128631 (plasmid)Phenylobacterium (2 proteins)DNA ligase CLSK2333706 Organism Protein name Accession J. FirmicutesBacteria; Firmicutes; Clostridia; Clostridiales; Peptococcaceae;Candidatus Desulforudis; Candidatus Desulforudis audaxviator CandidatusDesulforudis audaxviator ATP dependent YP_001716762 MP104CCandidatusDesulforudis (1 proteins) DNA ligase CLSK962101 Organism Protein nameAccession C. Actinobacteria Bacteria; Actinobacteria; Actinobacteria(class); Actinobacteridae; Actinomycetales; Micromonosporineae;Micromonosporaceae; Salinispora; Salinispora arenicola Salinisporaarenicola CNS-205Salinispora (2 proteins) DNA polymerase YP_001539124LigD ligase region Salinispora tropica CNB-440Salinispora (2 proteins)ATP dependent YP_001160776 DNA ligase CLSK915249 Organism Protein nameAccession C. Actinobacteria See CL5K2303611 above Bacteria;Actinobacteria; Actinobacteria (class); Actinobacteridae;Actinomycetales; Streptomycineae; Streptomycetaceae; Streptomyces;Streptomyces coelicolor Streptomyces avermitilis MA-4680 putative ATP-NP_828839 (plasmid)Streptomyces (2 proteins) dependint DNA ligaseStreptomyces sp. HK1 (plasmid)Streptomyces putative ATP- YP_001661618 (2proteins) dependent DNA ligase CLSK862724 Organism Protein nameAccession A. Archaea Archaea; Euryarchaeota; Archaeoglobi;Archaeoglobales; Archaeoglobaceae; Archaeoglobus; Archaeoglobus fulgidusArchaeoglobus fulgidus DSM 4304Archaeoglobus DNA ligase, NP_070553 (1proteins) putative J. Firmicutes Pelotomaculum thermopropionicumSIPelotomaculum ATP-dependent YP_001211793 (1 proteins) DNA ligaseThermoanaerobacter pseudethanolicus ATCC ATP dependent YP_00166447733223Thermoanaerobacter (2 proteins) DNA ligase CLSK820690 OrganismProtein name Accession A. Archaea Archaea; Euryarchaeota; environmentalsamples uncultured methanogenic archaeon RC-Ienvironmental ATP-dependentsamples (1 proteins) DNA ligase YP_686457 N. AlphaproteobacteriaBacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales;Bradyrhizobiaceae; Bradyrhizobium; Bradyrhizobium japonicumBradyrhizobium japonicum USDA 110Bradyrhizobium DNA ligase NP_774671 (2proteins) Bradyrhizobium sp. BTAilBradyrhizobium (2 dependentYP_001243518 proteins) putative ATP- DNA ligase CLSK808255 OrganismProtein name Accession N. Alphaproteobacteria Bacteria; Proteobacteria;Alphaproteobacteria; Rhizobiales; Rhizobiaceae; Sinorhizobium/Ensifergroup; Sinorhizobium; Sinorhizobium medicae Sinorhizobium medicaeWSM419Sinorhizobium DNA polymerase YP_001326990 (2 proteins) LigD ligaseregion Sinorhizobium meliloti 1021 (plasmid)Sinorhizobium putative ATP-NP_437750 (2 proteins) dependent DNA ligase protein CLSK806855 OrganismProtein name Accession N. Alphaproteobacteria Bacteria; Proteobacteria;Alphaproteobacteria; Rhizobiales; Rhizobiaceae; Rhizobium/Agrobacteriumgroup; Agrobacterium; Agrobacterium tumefaciens Agrobacteriumtumefaciens str. C58 ATP-dependent NP_396032 (plasmid)Agrobacterium (3proteins) DNA ligase Rhizobium leguminosarum bv. trifolii WSM1325 DNApolymerase YP_002973496 (plasmid)Rhizobium (10 proteins) domain LigD,ligase protein Rhizobium leguminosarum bv. trifolii WSM2304 DNApolymerase YP_002278005 (plasmid)Rhizobium (10 proteins) domain LigD,ligase protein CLSK390680 Organism Protein name Accession N.Alphaproteobacteria Bacteria; Proteobacteria; Alphaproteobacteria;Rhizobiales; Phyllobacteriaceae; Mesorhizobium; Mesorhizobium lotiMesorhizobium loti MAFF303099Mesorhizobium hypothetical NP_108282 (3proteins) protein

Additional exemplary SFLs may include, for example:

(SF DNA Ligase, GenBank ID AAC96909.1, fromParamecium bursaria Chlorella virus 1) (SEQ ID NO.: 77)MAITKPLLAATLENIEDVQFPCLATPKIDGIRSVKQTQMLSRTFKPIRNSVMNRLLTELLPEGSDGEISIEGATFQDTTSAVMTGHKMYNAKFSYYWFDYVTDDPLKKYIDRVEDMKNYITVHPHILEHAQVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGKVEEDVMGSIEVDYDGVVFSIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR;(MnM DNA Ligase, GenBank ID YP_333052.1, fromBurkholderia pseudomallei 1710b (equivalent sequence to ABA50091))(SEQ ID NO.: 78) MSGVPYGFKPNLAATLTKPELIKFPVWASPKIDGIRCVFFGGVAYSRSLKPIPNPVVQEFAKAYANLLEGLDGELTVGSPTDANCMQNSMAVMSKAAAPDFTFHVFDWFHPAQAHIEFWQRSDVVEDRIVQFYDRYPEVDIRAAPQVLCTSLAHLDTNEARWLADGYEGMMIRDHCGRYKFGRSTEREGGLVKVKRFTDAEAIVIGFEEEMHNANEAKRDATGRTERSTSKAGLHGKGTLGALVVKNERGIVFNIGTGFTAAQRADYWANHPSLFGKMVKFKHFDHGTVDAPRHPVFIGF RHPEDM;(Hin DNA Ligase, GenBank ID P44121, from Haemophilus influenza)(SEQ ID NO.: 79) MKFYRTLLLFFASSFAFANSDLMLLHTYNNQPIEGWVMSEKLDGVRGYWNGKQLLTRQGQRLSPPAYFIKDFPPFAIDGELFSERNHFEEIS T ITKSFKGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIPVKDKTHLYQFLAQVENLQGEGVVVRNPNAPYERKRSSQILKLKTAR G EECTVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSVITYKYR GITNSGKPRFATYWREKK;(DLX DNA Ligase, artificial ligase derived fromHin DNA ligase from Haemophilus influenza) (SEQ ID NO.: 80)MKFYRTLLLFFASSFAFANSDLMLLHTYNNQPIEGWVMSEKLDGVRGYWNGKQLLTRQGQRLSPPAYFIKDFPPFAIDGELFSERNHFEEIS S ITKSFKGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIPVKDKTHLYQFLAQVENLQGEGVVVRNPNAPYERKRSSQILKLKTARGEECTVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSVITYKYR GITNSGKPRFATYWREKK;(DLXd DNA Ligase, artificial ligase derived fromHin DNA ligase from Haemophilus influenza) (SEQ ID NO.: 81)MKFYRTLLLFFASSFAFANSDLMLLHTYNNQPIEGWVMSEKLDGVRGYWNGKQLLTRQGQRLSPPAYFIKDFPPFAIDGELFSERNHFEEIS S ITKSFKGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIPVKDKTHLYQFLAQVENLQGEGVVVRNPNAPYERKRSSQILKLKTAR D EECTVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSVITYKYR GITNSGKPRFATYWREKK;and, (DLXd2 DNA Ligase (Gammaproteobacteria,Haemophilus influenza) (modified)) (SEQ ID NO.: 82)MLLHTYNNQPIEGWVMSEKLDGVRGYWNGKQLLTRQGQRLSPPAYFIKDF PPFAIDGELFSERNHFEEISS ITKSFKGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIPVKDKTHLYQFLAQVENLQGEGVVVRN PNAPYERKRSSQILKLKTARD EECTVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSVITYKYRGITNSGKPRFATYWREKK.

As used herein, the terms “amplification”, “nucleic acid amplification”,or “amplifying” refer to the production of multiple copies of a nucleicacid template, or the production of multiple nucleic acid sequencecopies that are complementary to the nucleic acid template. Theamplification reaction may be a polymerase-mediated extension reactionsuch as, for example, a polymerase chain reaction (PCR). However, any ofthe known amplification reactions may be suitable for use as describedherein. The term “amplifying” that typically refers to an “exponential”increase in target nucleic acid may be used herein to describe bothlinear and exponential increases in the numbers of a select targetsequence of nucleic acid. The term “amplification reaction mixture”and/or “master mix” may refer to an aqueous solution comprising thevarious (some or all) reagents used to amplify a target nucleic acid.Such reactions may also be performed using solid supports (e.g., anarray). The reactions may also be performed in single or multiplexformat as desired by the user. These reactions typically includeenzymes, aqueous buffers, salts, amplification primers, target nucleicacid, and nucleoside triphosphates. Depending upon the context, themixture can be either a complete or incomplete amplification reactionmixture. The method used to amplify the target nucleic acid may be anyavailable to one of skill in the art. Any in vitro means for multiplyingthe copies of a target sequence of nucleic acid may be utilized. Theseinclude linear, logarithmic, and/or any other amplification method.While this disclosure may generally discuss PCR as the nucleic acidamplification reaction, it is expected that other types of nucleic acidamplification reactions, including both polymerase-mediatedamplification reactions (such as HDA, RPA, and RCA), as well asligase-mediated amplification reactions (such as LDR, LCR, andgap-versions of each), and combinations of nucleic acid amplificationreactions such as LDR and PCR (see for example U.S. Pat. No. 6,797,470)may also be suitable. For example, in addition to those describedelsewhere herein, various ligation-mediated reactions, where for exampleligation probes are employed as opposed to PCR primers. Additionalexemplary methods include polymerase chain reaction (PCR; see, e.g.,U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and/or 5,035,996),isothermal procedures (using one or more RNA polymerases (see, e.g., WO2006/081222), strand displacement (see, e.g., U.S. Pat. No. RE39007E),partial destruction of primer molecules (see, e.g., WO2006087574)),ligase chain reaction (LCR) (see, e.g., Wu, et al., Genomics 4: 560-569(1990)), and/or Barany, et al. PNAS USA 88:189-193 (1991)), Qβ RNAreplicase systems (see, e.g., WO/1994/016108), RNA transcription-basedsystems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g.,U.S. Pat. No. 5,854,033; U.S. Pub. No. 2004/265897; Lizardi et al. Nat.Genet. 19: 225-232 (1998); and/or Banér et al. Nucleic Acid Res., 26:5073-5078 (1998)), and strand displacement amplification (SDA) (Little,et al. Clin Chem 45:777-784 (1999)), among others. These systems, alongwith the many other systems available to the skilled artisan, may besuitable for use in amplifying target nucleic acids for use as describedherein.

“Amplification efficiency” may refer to any product that may bequantified to determine copy number (e.g., the term may refer to a PCRamplicon, an LCR ligation product, and/or similar product). Reactionsmay be compared by carrying out at least two separate amplificationreactions, each reaction being carried out in the absence and presence,respectively, of a reagent and/or step and quantifying amplificationthat occurs in each reaction.

Also provided are methods for amplifying a nucleic acid using at leastone polymerase, at least one primer, dNTPs, and ligating and amplifyingthe target nucleic acid. In some embodiments of such methods, at leastone primer is utilized. In certain embodiments, a nucleic acidamplification reaction mixture(s) comprising at least one polymerase,dNTPs, and at least one primer is provided. In other embodiments,methods for using such mixture(s) are provided. Target nucleic acids maybe amplified using any of a variety of reactions and systems. Exemplarymethods for amplifying nucleic acids include, for example,polymerase-mediated extension reactions. For instance, thepolymerase-mediated extension reaction can be the polymerase chainreaction (PCR). In other embodiments, the nucleic acid amplificationreaction is a multiplex reaction. For instance, exemplary methods foramplifying and detecting nucleic acids suitable for use as describedherein are commercially available as TaqMan® (see, e.g., U.S. Pat. Nos.4,889,818; 5,079,352; 5,210,015; 5,436,134; 5,487,972; 5,658,751;5,210,015; 5,487,972; 5,538,848; 5,618,711; 5,677,152; 5,723,591;5,773,258; 5,789,224; 5,801,155; 5,804,375; 5,876,930; 5,994,056;6,030,787; 6,084,102; 6,127,155; 6,171,785; 6,214,979; 6,258,569;6,814,934; 6,821,727; 7,141,377; and/or 7,445,900, all of which arehereby incorporated herein by reference in their entirety). TaqMan®assays are typically carried out by performing nucleic acidamplification on a target polynucleotide using a nucleic acid polymerasehaving 5′-3′ nuclease activity, a primer capable of hybridizing to saidtarget polynucleotide, and an oligonucleotide probe capable ofhybridizing to said target polynucleotide 3′ relative to said primer. Insome embodiments, the oligonucleotide probe includes a detectable label(e.g., a fluorescent reporter molecule) and a quencher molecule capableof quenching the fluorescence of said reporter molecule. In certainembodiments, the detectable label and quencher molecule are part of asingle probe. As amplification proceeds, the polymerase digests theprobe to separate the detectable label from the quencher molecule. Thedetectable label (e.g., fluorescence) can be monitored during thereaction, where detection of the label corresponds to the occurrence ofnucleic acid amplification (e.g., the higher the signal the greater theamount of amplification). Variations of TaqMan® assays (e.g., LNA™spiked TaqMan® assay) are known in the art and would be suitable for usein the methods described herein.

Another exemplary system suitable for use as described herein utilizesdouble-stranded probes in displacement hybridization methods (see, e.g.,Morrison et al. Anal. Biochem., 18:231-244 (1989); and/or Li, et al.Nucleic Acids Res., 30(2,e5) (2002)). In such methods, the probetypically includes two complementary oligonucleotides of differentlengths where one includes a detectable label and the other includes aquencher molecule. When not bound to a target nucleic acid, the quenchersuppresses the signal from the detectable label. The probe becomesdetectable upon displacement hybridization with a target nucleic acid.Multiple probes may be used, each containing different detectablelabels, such that multiple target nucleic acids may be queried in asingle reaction.

Additional exemplary methods for amplifying and detecting target nucleicacids suitable for use as described herein involve “molecular beacons”,which are single-stranded hairpin shaped oligonucleotide probes. In thepresence of the target sequence, the probe unfolds, binds and emits asignal (e.g., fluoresces). A molecular beacon typically includes atleast four components: 1) the “loop”, an 18-30 nucleotide region whichis complementary to the target sequence; 2) two 5-7 nucleotide “stems”found on either end of the loop and being complementary to one another;3) at the 5′ end, a detectable label; and 4) at the 3′ end, a quencherdye that prevents the detectable label from emitting a single when theprobe is in the closed loop shape (e.g., not bound to a target nucleicacid). Thus, in the presence of a complementary target, the “stem”portion of the beacon separates out resulting in the probe hybridizingto the target. Other types of molecular beacons are also known and maybe suitable for use in the methods described herein. Molecular beaconsmay be used in a variety of assay systems. One such system is nucleicacid sequence-based amplification (NASBA®), a single step isothermalprocess for amplifying RNA to double stranded DNA without temperaturecycling. A NASBA reaction typically requires avian myeloblastosis virus(AMV), reverse transcriptase (RT), T7 RNA polymerase, RNase H, and twooligonucleotide primers. After amplification, the amplified targetnucleic acid may be detected using a molecular beacon. Other uses formolecular beacons are known in the art and would be suitable for use inthe methods described herein.

The Scorpion system is another exemplary assay format that may be usedin the methods described herein. Scorpion primers are bi-functionalmolecules in which a primer is covalently linked to the probe, alongwith a detectable label (e.g., a fluorophore) and a quencher. In thepresence of a target nucleic acid, the detectable label and the quencherseparate which leads to an increase in signal emitted from thedetectable label. Typically, a primer used in the amplification reactionincludes a probe element at the 5′ end along with a “PCR blocker”element (e.g., a hexethylene glycol (HEG) monomer (Whitcombe, et al.Nat. Biotech. 17: 804-807 (1999)) at the start of the hairpin loop. Theprobe typically includes a self-complementary stem sequence with adetectable label at one end and a quencher at the other. In the initialamplification cycles (e.g., PCR), the primer hybridizes to the targetand extension occurs due to the action of polymerase. The Scorpionsystem may be used to examine and identify point mutations usingmultiple probes that may be differently tagged to distinguish betweenthe probes. Using PCR as an example, after one extension cycle iscomplete, the newly synthesized target region will be attached to thesame strand as the probe. Following the second cycle of denaturation andannealing, the probe and the target hybridize. The hairpin sequence thenhybridizes to a part of the newly produced PCR product. This results inthe separation of the detectable label from the quencher and causesemission of the signal. Other uses for molecular beacons are known inthe art and would be suitable for use in the methods described herein.

The nucleic acid polymerases that may be employed in the disclosednucleic acid amplification reactions may be any that function to carryout the desired reaction including, for example, a prokaryotic, fungal,viral, bacteriophage, plant, and/or eukaryotic nucleic acid polymerase.As used herein, the term “DNA polymerase” refers to an enzyme thatsynthesizes a DNA strand de novo using a nucleic acid strand as atemplate. DNA polymerase uses an existing DNA or RNA as the template forDNA synthesis and catalyzes the polymerization of deoxyribonucleotidesalongside the template strand, which it reads. The newly synthesized DNAstrand is complementary to the template strand. DNA polymerase can addfree nucleotides only to the 3′-hydroxyl end of the newly formingstrand. It synthesizes oligonucleotides via transfer of a nucleosidemonophosphate from a deoxyribonucleoside triphosphate (dNTP) to the3′-hydroxyl group of a growing oligonucleotide chain. This results inelongation of the new strand in a 5′ to 3′ direction. Since DNApolymerase can only add a nucleotide onto a pre-existing 3′-OH group, tobegin a DNA synthesis reaction, the DNA polymerase needs a primer towhich it can add the first nucleotide. Suitable primers may compriseoligonucleotides of RNA or DNA, or chimeras thereof (e.g., RNA/DNAchimerical primers). The DNA polymerases may be a naturally occurringDNA polymerases or a variant of natural enzyme having theabove-mentioned activity. For example, it may include a DNA polymerasehaving a strand displacement activity, a DNA polymerase lacking 5′ to 3′exonuclease activity, a DNA polymerase having a reverse transcriptaseactivity, or a DNA polymerase having an endonuclease activity.

Suitable nucleic acid polymerases may also comprise holoenzymes,functional portions of the holoenzymes, chimeric polymerase, or anymodified polymerase that can effectuate the synthesis of a nucleic acidmolecule. Within this disclosure, a DNA polymerase may also include apolymerase, terminal transferase, reverse transcriptase, telomerase,and/or polynucleotide phosphorylase. Non-limiting examples ofpolymerases may include, for example, T7 DNA polymerase, eukaryoticmitochondrial DNA Polymerase γ, prokaryotic DNA polymerase I, II, III,IV, and/or V; eukaryotic polymerase α, β, γ, δ, ε, η, ζ, ι, and/or κ; E.coli DNA polymerase I; E. coli DNA polymerase III alpha and/or epsilonsubunits; E. coli polymerase IV, E. coli polymerase V; T. aquaticus DNApolymerase I; B. stearothermophilus DNA polymerase I; Euryarchaeotapolymerases; terminal deoxynucleotidyl transferase (TdT); S. cerevisiaepolymerase 4; translesion synthesis polymerases; reverse transcriptase;and/or telomerase. Non-limiting examples of suitable thermostable DNApolymerases that may be used include Taq, Tfl, Pfu, and Vent™ DNApolymerases, any genetically engineered DNA polymerases, any havingreduced or insignificant 3′ to 5′ exonuclease activity (e.g.,SuperScript™ DNA polymerase), and/or genetically engineered DNApolymerases (e.g., those having the active site mutation F667Y or theequivalent of F667Y (e.g., in Tth), AmpliTaqFS, ThermoSequenase™),Therminator I, Therminator II, Therminator III, Therminator Gamma (allavailable from NEB), and/or any derivatives and fragments thereof. Othernucleic acid polymerases may also be suitable as would be understood byone of skill in the art.

In another aspect, the present disclosure provides reaction mixtures foramplifying a nucleic acid sequence of interest (e.g., a targetsequence). In some embodiments, the reaction mixture may furthercomprise a signal-generating compound (SGC) and/or detectable label. Themethods may also include one or more steps for detecting the SGC and/ordetectable label to quantitate the amplified nucleic acid.

A SGC may be a substance that is itself detectable in an assay ofchoice, or capable of reacting to form a chemical or physical entity(e.g., a reaction product) that is detectable in an assay of choice.Representative examples of reaction products include precipitates,fluorescent signals, compounds having a color, and the like.Representative SGC include e.g., bioluminescent compounds (e.g.,luciferase), fluorophores (e.g., below), bioluminescent andchemiluminescent compounds, radioisotopes (e.g., ¹³¹I, ¹²⁵I, ¹⁴C, ³H,³⁵S, ³²P and the like), enzymes (e.g., below), binding proteins (e.g.,biotin, avidin, streptavidin and the like), magnetic particles,chemically reactive compounds (e.g., colored stains), labeledoligonucleotides; molecular probes (e.g., CY3, Research Organics, Inc.),and the like. Representative fluorophores include fluoresceinisothiocyanate, succinyl fluorescein, rhodamine B, lissamine,9,10-diphenlyanthracene, perylene, rubrene, pyrene and fluorescentderivatives thereof such as isocyanate, isothiocyanate, acid chloride orsulfonyl chloride, umbelliferone, rare earth chelates of lanthanidessuch as Europium (Eu) and the like. Representative SGC's useful in asignal generating conjugate include the enzymes in: TUB Class 1,especially 1.1.1 and 1.6 (e.g., alcohol dehydrogenase, glyceroldehydrogenase, lactate dehydrogenase, malate dehydrogenase,glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphatedehydrogenase and the like); IUB Class 1.11.1 (e.g., catalase,peroxidase, amino acid oxidase, galactose oxidase, glucose oxidase,ascorbate oxidase, diaphorase, urease and the like); TUB Class 2,especially 2.7 and 2.7.1 (e.g., hexokinase and the like); TUB Class 3,especially 3.2.1 and 3.1.3 (e.g., alpha amylase, cellulase,β-galacturonidase, amyloglucosidase, β-glucuronidase, alkalinephosphatase, acid phosphatase and the like); TUB Class 4 (e.g., lyases);TUB Class 5 especially 5.3 and 5.4 (e.g., phosphoglucose isomerase,trios phosphatase isomerase, phosphoglucose mutase and the like.) SGCsmay also generate products detectable by fluorescent andchemiluminescent wavelengths, e.g., sequencing dyes, luciferase,fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanideseries; compounds such as luminol, isoluminol, acridinium salts, and thelike; bioluminescent compounds such as luciferin; fluorescent proteins(e.g., GFP or variants thereof); and the like. Attaching certain SGC toagents can be accomplished through metal chelating groups such as EDTA.The subject SGC shares the common property of allowing detection and/orquantification of an attached molecule. SGCs are optionally detectableusing a visual or optical method; preferably, with a method amenable toautomation such as a spectrophotometric method, a fluorescence method, achemiluminescent method, an electrical nanometric method involving e.g.,a change in conductance, impedance, resistance and the like and amagnetic field method. Some SGCs are optionally detectable with thenaked eye or with a signal detection apparatus. Some SGCs are notthemselves detectable but become detectable when subject to furthertreatment. The SGC can be attached in any manner (e.g., through covalentor non-covalent bonds) to a binding agent of interest (e.g., an antibodyor a PDZ polypeptide). SGCs suitable for attachment to agents such asantibodies include colloidal gold, fluorescent antibodies, Europium,latex particles, and enzymes. The agents that bind to NS1 and NP caneach comprise distinct SGCs. For example, red latex particles can beconjugated to anti-NS1 antibodies and blue latex particles can beconjugated to anti-NP antibodies. Other detectable SGCs suitable for usein a lateral flow format include any moiety that is detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical, chemical, or other means. For example, suitable SGCs includebiotin for staining with labeled streptavidin conjugate, fluorescentdyes (e.g., fluorescein, Texas red, rhodamine, green fluorescentprotein, and the like), radiolabels, enzymes (e.g., horseradishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric SGCs such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex beads). Patents that describedthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. See alsoHandbook of Fluorescent Probes and Research Chemicals (6th Ed.,Molecular Probes, Inc., Eugene, Oreg.). Radiolabels can be detectedusing photographic film or scintillation counters, fluorescent markerscan be detected using a photodetector to detect emitted light.

Similarly, the term “detectable label” may refer to any of a variety ofsignaling molecules indicative of amplification. For example, SYBR GREENand other DNA-binding dyes are detectable labels. Such detectable labelsmay comprise or may be, for example, nucleic acid intercalating agentsor non-intercalating agents. As used herein, an intercalating agent isan agent or moiety capable of non-covalent insertion between stackedbase pairs of a double-stranded nucleic acid molecule. Anon-intercalating agent is one that does not insert into thedouble-stranded nucleic acid molecule. The nucleic acid binding agentmay produce a detectable signal directly or indirectly. The signal maybe detectable directly using, for example, fluorescence and/orabsorbance, or indirectly using, for example, any moiety or ligand thatis detectably affected by proximity to double-stranded nucleic acid issuitable such as a substituted label moiety or binding ligand attachedto the nucleic acid binding agent. It is typically necessary for thenucleic acid binding agent to produce a detectable signal when bound toa double-stranded nucleic acid that is distinguishable from the signalproduced when that same agent is in solution or bound to asingle-stranded nucleic acid. For example, intercalating agents such asethidium bromide fluoresce more intensely when intercalated intodouble-stranded DNA than when bound to single-stranded DNA, RNA, or insolution (see, e.g., U.S. Pat. Nos. 5,994,056; 6,171,785; and/or6,814,934). Similarly, actinomycin D fluoresces red fluorescence whenbound to single-stranded nucleic acids, and green when bound todouble-stranded nucleic acids. And in another example, the photoreactivepsoralen 4-aminomethyle-4-5′8-trimethylpsoralen (AMT) has been reportedto exhibit decreased absorption at long wavelengths and fluorescenceupon intercalation into double-stranded DNA (Johnson et al. Photochem. &Photobiol., 33:785-791 (1981). For example, U.S. Pat. No. 4,257,774describes the direct binding of fluorescent intercalators to DNA (e.g.,ethidium salts, daunomycin, mepacrine and acridine orange,4′6-diamidino-α-phenylindole). Non-intercalating agents (e.g., minorgroove binders as described herein such as Hoechst 33258, distamycin,netropsin) may also be suitable for use. For example, Hoechst 33258(Searle, et al. Nuc. Acids Res. 18(13):3753-3762 (1990)) exhibitsaltered fluorescence with an increasing amount of target. Minor groovebinders are described in more detail elsewhere herein.

Other DNA binding dyes are available to one of skill in the art and maybe used alone or in combination with other agents and/or components ofan assay system. Exemplary DNA binding dyes may include, for example,acridines (e.g., acridine orange, acriflavine), actinomycin D (Jain, etal. J. Mol. Biol. 68:21 (1972)), anthramycin, BOBO™-1, BOBO™-3,BO-PRO™-1, cbromomycin, DAPI (Kapuseinski, et al. Nuc. Acids Res.6(112): 3519 (1979)), daunomycin, distamycin (e.g., distamycin D), dyesdescribed in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts (e.g.,ethidium bromide), fluorcoumanin, fluorescent intercalators as describedin U.S. Pat. No. 4,257,774, GelStar® (Cambrex Bio Science Rockland Inc.,Rockland, Me.), Hoechst 33258 (Searle and Embrey, 1990, Nuc. Acids Res.18:3753-3762), Hoechst 33342, homidium, JO-PRO™-1, LIZ dyes, LO-PRO™-1,mepacrine, mithramycin, NED dyes, netropsin,4′6-diamidino-α-phenylindole, proflavine, POPO™-1, POPO™-3, PO-PRO™-1,propidium iodide, ruthenium polypyridyls, S5, SYBR® Gold, SYBR® Green I(U.S. Pat. Nos. 5,436,134 and 5,658,751), SYBR® Green II, SYTOX blue,SYTOX green, SYTO® 43, SYTO® 44, SYTO® 45, SYTOX® Blue, TO-PRO®-1, SYTO®11, SYTO® 13, SYTO® 15, SYTO® 16, SYTO® 20, SYTO® 23, thiazole orange(Aldrich Chemical Co., Milwaukee, Wis.), TOTO™-3, YO-PRO®-1, and YOYO®-3(Molecular Probes, Inc., Eugene, Oreg.), among others. SYBR® Green I(see, e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and/or 6,569,927), forexample, has been used to monitor a PCR reactions. Other DNA bindingdyes may also be suitable as would be understood by one of skill in theart.

For use as described herein, one or more detectable labels and/orquenching agents may be attached to one or more primers and/or probes(e.g., detectable label). The detectable label may emit a signal whenfree or when bound to one of the target nucleic acids. The detectablelabel may also emit a signal when in proximity to another detectablelabel. Detectable labels may also be used with quencher molecules suchthat the signal is only detectable when not in sufficiently closeproximity to the quencher molecule. For instance, in some embodiments,the assay system may cause the detectable label to be liberated from thequenching molecule. Any of several detectable labels may be used tolabel the primers and probes used in the methods described herein. Asmentioned above, in some embodiments the detectable label may beattached to a probe, which may be incorporated into a primer, or mayotherwise bind to the amplified target nucleic acid (e.g., a detectablenucleic acid binding agent such as an intercalating or non-intercalatingdye). When using more than one detectable label, each should differ intheir spectral properties such that the labels may be distinguished fromeach other, or such that together the detectable labels emit a signalthat is not emitted by either detectable label alone. Exemplarydetectable labels include, for instance, a fluorescent dye or fluorphore(e.g., a chemical group that can be excited by light to emitfluorescence or phosphorescence), “acceptor dyes” capable of quenching afluorescent signal from a fluorescent donor dye, and the like. Suitabledetectable labels may include, for example, fluorosceins (e.g.,5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT(Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE;6-carboxyfluorescein (6-FAM); FITC;6-carboxy-1,4-dichloro-2′,7′-dichlorofluorescein (TET);6-carboxy-1,4-dichloro-2′,4′, 5′, 7′-tetrachlorofluorescein (HEX);6-carboxy-4′,5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE);); Alexafluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594,610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g.,492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570,576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP,FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S,AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin,CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes(e.g., calcium crimson, calcium green, calcium orange, calcofluorwhite), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5,5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescentproteins (e.g., green fluorescent protein (e.g., GFP. EGFP), bluefluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1), cyanfluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescentprotein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs(e.g., fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,EDANS/dabcyl, fluorescein/fluorescein, BODIPY FL/BODIPY FL,Fluorescein/QSY7 and QSY9), LysoTracker and LysoSensor (e.g.,LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker YellowHCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensorBlue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153,LysoSensor Yellow/Blue DND-160, LysoSensor Yellow/Blue 10,000 MWdextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g.,110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine(5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine RhodamineB, Phallicidine, Phalloidine, Red, Rhod-2, ROX (6-carboxy-X-rhodamine),5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine GExtra, TAMRA (6-carboxytetramethylrhodamine), Tetramethylrhodamine(TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in,e.g., U.S. Pub. No. 2009/0197254 (incorporated herein by reference inits entirety), among others as would be known to those of skill in theart. Other detectable labels may also be used (see, e.g., U.S. Pub. No.2009/0197254 (incorporated herein by reference in its entirety)), aswould be known to those of skill in the art. Any of these systems anddetectable labels, as well as many others, may be used to detectamplified target nucleic acids.

Some detectable labels can be sequence-based (also referred to herein asa “locus-specific detectable label”), for example 5′ nuclease probes.Such probes may comprise one or more detectable labels. Variousdetectable labels are known in the art, for example (TaqMan® probesdescribed herein (See also U.S. Pat. No. 5,538,848 (incorporated hereinby reference in its entirety)) various stem-loop molecular beacons (See,e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996,Nature Biotechnology 14:303-308), stemless or linear beacons (See, e.g.,WO 99/21881; U.S. Pat. No. 6,485,901), PNA Molecular Beacons™ (See,e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (See,e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (See,e.g., U.S. Pat. No. 6,150,097), Sunrise®/Amplifluor® probes (U.S. Pat.No. 6,548,250), stem-loop and duplex Scorpion™ probes (Solinas et al.,2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulgeloop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No.6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe(Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptidenucleic acid (PNA) light-up probes (Svanvik, et al. Anal Biochem281:26-35 (2001)), self-assembled nanoparticle probes,ferrocene-modified probes described, for example, in U.S. Pat. No.6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al.,1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, MolecularCell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35;Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002,Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, NucleicAcids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332;Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al.,2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res.Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc14:11155-11161; QuantiProbes (www.qiagen.com), HyBeacons (French, et al.Mol. Cell. Probes 15:363-374 (2001)), displacement probes (Li, et al.Nucliec Acids Res. 30:e5 (2002)), HybProbes (Cardullo, et al. PNAS85:8790-8794 (1988)), MGB Alert (www.nanogen.com), Q-PNA (Fiandaca, etal. Genome Res. 11:609-611 (2001)), Plexor (www.Promega.com), LUXprimers (Nazarenko, et al. Nucleic Acids Res. 30:e37 (2002)), DzyNAprimers (Todd, et al. Clin. Chem. 46:625-630 (2000)). Detectable labelscan also comprise black hole quenchers (Biosearch), Iowa Black (IDT),QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate/carboxylate Quenchers (Epoch). Detectable labels can alsocomprise two probes, wherein for example a fluor is on one probe, and aquencher on the other, wherein hybridization of the two probes togetheron a target quenches the signal, or wherein hybridization on a targetalters the signal signature via a change in fluorescence. Exemplarysystems may also include FRET, salicylate/DTPA ligand systems (see,e.g., Oser et al. Angew. Chem. Int. Engl. 29(10):1167 (1990)),displacement hybridization, homologous probes, and/or assays describedin EP 070685 and/or U.S. Pat. No. 6,238,927. Detectable labels can alsocomprise sulfonate derivatives of fluorescein dyes with SO₃ instead ofthe carboxylate group, phosphoramidite forms of fluorescein,phosphoramidite forms of CY5 (available for example from Amersham).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues, orany variant or functional fragment thereof. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. The term “amino acid” includes naturallyoccurring and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, e.g., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. The following eight groups eachcontain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); [0078] 4) Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) [0083] (see, e.g.,Creighton, Proteins (1984)). Variants of a given nucleotide sequence orpolypeptide sequence are optionally conservatively modified variants.With respect to particular nucleic acid sequences, conservativelymodified variants refers to those nucleic acids which encode identicalor essentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences.

The term “antibody” or “antibodies” may include whole and/or fragmentsand/or derivatives of antibodies in unpurified or partially purifiedform (e.g., hybridoma supernatant, ascites, polyclonal antisera) or inpurified form. A “purified” antibody may be one that is separated fromat least about 50% of the proteins with which it is initially found(e.g., as part of a hybridoma supernatant or ascites preparation).Preferably, a purified antibody is separated from at least about 60%,75%, 90%, or 95% of the proteins with which it is initially found.Suitable derivatives may include fragments (e.g., Fab, Fab₂ or singlechain antibodies (Fv for example)), as are known in the art. Theantibodies may be of any suitable origin or form including, for example,murine (e.g., produced by murine hybridoma cells), or expressed ashumanized antibodies, chimeric antibodies, human antibodies, and thelike. Methods of preparing and utilizing various types of antibodies arewell-known to those of skill in the art and would be suitable for use(see, for example, Harlow, et al. Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: ALaboratory Manual, Portable Protocol No. 1, 1998; Kohler and Milstein,Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986);Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct.Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536(1988); Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al.,J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992);Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13(1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos.4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and,5,661,016).

In certain applications, the antibodies may be contained withinhybridoma supernatant or ascites and utilized either directly as such orfollowing concentration using standard techniques. In otherapplications, the antibodies may be further purified using, for example,salt fractionation and ion exchange chromatography, or affinitychromatography using Protein A, Protein G, Protein A/G, and/or Protein Lligands covalently coupled to a solid support such as agarose beads, orcombinations of these techniques. The antibodies may be stored in anysuitable format, including as a frozen preparation (e.g., about −20° C.or −70° C.), in lyophilized form, or under normal refrigerationconditions (e.g., about 4° C.). When stored in liquid form, it ispreferred that a suitable buffer such as Tris-buffered saline (TBS) orphosphate buffered saline (PBS) is utilized. Antibodies and theirderivatives may be incorporated into compositions (e.g., attached tooligonucleotides) described herein for use in vitro or in vivo.Antibodies may also be modified for use by, for example, biotinylation.Other methods for making and using antibodies available to one of skillin the art may also be suitable for use.

The methods described herein may be useful for detecting and/orquantifying a variety of target nucleic acids from a test sample (e.g.,biological sample). A target nucleic acid is any nucleic acid for whichan assay system is designed to identify or detect as present (or not),and/or quantify in a test sample. Such nucleic acids may include, forexample, those of infectious agents (e.g., virus, bacteria, parasite,and the like), a disease process such as cancer, diabetes, or the like,or to measure an immune response. Exemplary “test samples” includevarious types of samples, such as biological samples. Exemplarybiological samples include, for instance, a bodily fluid (e.g., blood,saliva, spinal fluid), a tissue sample, a food (e.g., meat) or beverage(e.g., milk) product, or the like. Other examples of biological samplesmay include, whole blood, serum, plasma, urine, synovial fluid, saliva,cerebrospinal fluid, tissue infiltrate, cervical or vaginal exudate,pleural effusion, bronchioalveolar lavage fluid, gastric lavage fluid,small or large bowel contents, and swab specimens from various bodilyorifices dispersed in a suitable medium. Expressed nucleic acids mayinclude, for example, genes for which expression (or lack thereof) isassociated with medical conditions such as infectious disease (e.g.,bacterial, viral, fungal, protozoal infections) or cancer. The methodsdescribed herein may also be used to detect contaminants (e.g.,bacteria, virus, fungus, and/or protozoan) in pharmaceutical, food, orbeverage products. The methods described herein may be also be used todetect rare alleles in the presence of wild type alleles (e.g., onemutant allele in the presence of 10⁶-10⁹ wild type alleles). The methodsare useful to, for example, detect minimal residual disease (e.g., rareremaining cancer cells during remission, especially mutations in the p53gene or other tumor suppressor genes previously identified within thetumors), and/or measure mutation load (e.g., the frequency of specificsomatic mutations present in normal tissues, such as blood or urine).

Kits for performing the methods described herein are also provided. Thekit may comprise one or more probes (e.g., antibody conjugated to anoligonucleotide) a pair of oligonucleotides for amplifying at least onetarget nucleic acid from a sample, a biocatalyst (e.g., DNA polymerase)and/or corresponding one or more probes labeled with a detectable label.The kit may also include samples containing pre-defined target nucleicacids to be used in control reactions. The kit may also optionallyinclude stock solutions, buffers, enzymes, detectable labels or reagentsrequired for detection, tubes, membranes, and the like that may be usedto complete the amplification reaction. In some embodiments, multipleprimer sets are included. Other embodiments of particular systems andkits are also contemplated which would be understood by one of skill inthe art.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to this Description.

Unless otherwise apparent from the context, any feature can be claimedin combination with any other, or be claimed as not present incombination with another feature. A feature can be any piece ofinformation that can characterize an invention or can limit the scope ofa claim, for example any variation, step, feature, property,composition, method, step, degree, level, component, material,substance, element, mode, variable, aspect, measure, amount, option,embodiment, clause, descriptive term, claim element or limitation.

The singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. Where necessary, ranges havebeen supplied, and those ranges are inclusive of all sub-ranges therebetween.

In this disclosure, the use of the singular can include the pluralunless specifically stated otherwise or unless, as will be understood byone of skill in the art in light of the present disclosure, the singularis the only functional embodiment. Thus, for example, “a” may mean morethan one, and “one embodiment” may mean that the description applies tomultiple embodiments. The phrase “and/or” denotes a shorthand way ofindicating that the specific combination is contemplated in combinationand, separately, in the alternative.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc. discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. Also, the use of “comprise,”“comprises,” “comprising,” “contain,” “contains,” “containing,”“include,” “includes,” and “including” are not intended to be limiting.It is to be understood that both the foregoing general description anddetailed description are exemplary and explanatory only and are notrestrictive of the invention.

Unless specifically noted in the above specification, embodiments in theabove specification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims).

Generally, features described herein are intended to be optional unlessexplicitly indicated to be necessary in the specification. Non-limitingexamples of language indicating that a feature is regarded as optionalin the specification include terms such as “variation,” “where,”“while,” “when,” “optionally,” “include,” “preferred,” “especial,”“recommended,” “advisable,” “particular,” “should,” “alternative,”“typical,” “representative,” “various,” “such as,” “the like,” “can,”“may,” “example,” “embodiment,” or “aspect,” “in some,” “example,”“exemplary,” “instance,” “if” or any combination and/or variation ofsuch terms.

“Isolated” or “purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises a significant percent(e.g., greater than 2%, greater than 5%, greater than 10%, greater than20%, greater than 50%, or more, sometimes more than 90%, 95% or 99%) ofthe sample in which it resides. In certain embodiments, a substantiallypurified component comprises at least 50%, 80%-85%, or 90-95% of thesample. Techniques for purifying polynucleotides and polypeptides ofinterest are well-known in the art and include, for example,ion-exchange chromatography, affinity chromatography and sedimentationaccording to density. Generally, a substance is purified when it existsin a sample in a higher proportion than it is naturally found.

Sequence identity (also called homology) refers to similarity insequence of two or more sequences (e.g., nucleotide or polypeptidesequences). In the context of two or more homologous sequences, thepercent identity or homology of the sequences or subsequences thereofindicates the percentage of all monomeric units (e.g., nucleotides oramino acids) that are the same (e.g., about 70% identity, preferably75%, 80%, 85%, 90%, 95% or 99% identity). The percent identity can beover a specified region, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection. Sequences are said to be “substantially identical” whenthere is at least 90% identity at the amino acid level or at thenucleotide level. This definition also refers to the complement of atest sequence. Preferably, the identity exists over a region that is atleast about 25, 50, or 100 residues in length, or across the entirelength of at least one compared sequence. A preferred algorithm fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al,Nuc. Acids Res. 25:3389-3402 (1977). Other methods include thealgorithms of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), andNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc. Another indicationthat two nucleic acid sequences are substantially identical is that thetwo molecules or their complements hybridize to each other understringent conditions.

Any indication that a feature is optional is intended provide adequatesupport (e.g., under 35 U.S.C. 112 or Art. 83 and 84 of EPC) for claimsthat include closed or exclusive or negative language with reference tothe optional feature. Exclusive language specifically excludes theparticular recited feature from including any additional subject matter.For example, if it is indicated that A can be drug X, such language isintended to provide support for a claim that explicitly specifies that Aconsists of X alone, or that A does not include any other drugs besidesX. “Negative” language explicitly excludes the optional feature itselffrom the scope of the claims. For example, if it is indicated thatelement A can include X, such language is intended to provide supportfor a claim that explicitly specifies that A does not include X.Non-limiting examples of exclusive or negative terms include “only,”“solely,” “consisting of,” “consisting essentially of,” “alone,”“without”, “in the absence of (e.g., other items of the same type,structure and/or function)” “excluding,” “not including”, “not”,“cannot,” or any combination and/or variation of such language.

Similarly, referents such as “a,” “an,” “said,” or “the,” are intendedto support both single and/or plural occurrences unless the contextindicates otherwise. For example “a dog” is intended to include supportfor one dog, no more than one dog, at least one dog, a plurality ofdogs, etc. Non-limiting examples of qualifying terms that indicatesingularity include “a single”, “one,” “alone,” “only one,” “not morethan one,” etc. Non-limiting examples of qualifying terms that indicate(potential or actual) plurality include “at least one,” “one or more,”“more than one,” “two or more,” “a multiplicity,” “a plurality,” “anycombination of,” “any permutation of,” “any one or more of,” etc. Claimsor descriptions that include “or” between one or more members of a groupare considered satisfied if one, more than one, or all of the groupmembers are present in, employed in, or otherwise relevant to a givenproduct or process unless indicated to the contrary or otherwise evidentfrom the context.

In the claims, any active verb (or its gerund) is intended to indicatethe corresponding actual or attempted action, even if no actual actionoccurs. For example, the verb “hybridize” and gerund form “hybridizing”and the like refer to actual hybridization or to attempted hybridizationby contacting nucleic acid sequences under conditions suitable forhybridization, even if no actual hybridization occurs. Similarly,“detecting” and “detection” when used in the claims refer to actualdetection or to attempted detection, even if no target is actuallydetected.

Furthermore, it is to be understood that the inventions encompass allvariations, combinations, and permutations of any one or more featuresdescribed herein. Any one or more features may be explicitly excludedfrom the claims even if the specific exclusion is not set forthexplicitly herein. It should also be understood that disclosure of areagent for use in a method is intended to be synonymous with (andprovide support for) that method involving the use of that reagent,according either to the specific methods disclosed herein, or othermethods known in the art unless one of ordinary skill in the art wouldunderstand otherwise. In addition, where the specification and/or claimsdisclose a method, any one or more of the reagents disclosed herein maybe used in the method, unless one of ordinary skill in the art wouldunderstand otherwise.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety into this application as ifeach individual publication or patent were specifically and individuallyindicated to be incorporated by reference. Genbank records referenced byGID or accession number, particularly any polypeptide sequence,polynucleotide sequences or annotation thereof, are incorporated byreference herein. The citation of any publication is for its disclosureprior to the filing date and should not be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention.

Where ranges are given herein, the endpoints are included. Furthermore,it is to be understood that unless otherwise indicated or otherwiseevident from the context and understanding of one of ordinary skill inthe art, values that are expressed as ranges can assume any specificvalue or subrange within the stated ranges in different embodiments ofthe invention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

Certain embodiments are further described in the following examples.These embodiments are provided as examples only and are not intended tolimit the scope of the claims in any way.

EXAMPLES Example 1

In an exemplary embodiment of typical proximity ligation assay (PLA)processes (FIG. 1), the probe mix (e.g., comprising two probes, A and B,(each probe comprising a streptavidin oligo “SAO” component and anantibody “Ab” component) in probe dillution buffer “PDB”) and testsample (in sample dillution buffer “SDB”) are combined into a bindingreaction. Following the binding reaction (e.g., at 37° C. for 1 hour),the ligation reaction mixture is added in order to carry out theligation reaction. To prepare the ligation reaction mixture, the ligaseand ligation buffer are diluted. Following the ligation reaction (e.g.,at 37° C. for 10 minutes), the ligated product is stabilized by proteasedigestion; the protease is then inactivated (e.g., using heat byincubation at 37° C. for 10 minutes followed by 95° C. for 5 minutes).Usually, a portion of the ligated product is transferred to thereal-time PCR reaction mixture (comprising PCR primers and proximityprobe mix “PCR-PP”), then placed on the PCR reaction plate in a qPCRinstrument. Detection and quantification of the ligated product can thenproceed using standard techniques.

A schematic of an exemplary improved PLA process is illustrated in FIG.2. As shown therein, the binding reaction is the same as shown inFIG. 1. However, in some embodiments of the improved processes disclosedherein, as shown in FIG. 2, ligase is added to a real-time PCR mixture(comprising PCR primers, proximity probe and splint mix “PCR-PPS”) whichis then added directly to the binding reaction. In certain embodimentsof the improved PLA processes, a test sample (e.g., cell lysate) isprepared, a binding reaction is allowed to take place and then aligation buffer added directly thereto. To that mixture is then added aproximity probe mixture, and a PCR mixture. This combined reactionmixture is then incubated for a suitable amount of time (e.g., roomtemperature for 20 minutes and then 96° C. for 5 min) and PCR isperformed. The PCR reaction mixture is then deposited onto the reactionplate in a qPCR instrument and detection and quantification of theligated product can then proceed using standard techniques (as intypical PLA processes).

Example 2

Some exemplary embodiments of typical and improved PLA processes arealso compared in FIGS. 3A and 3B. As shown in the embodimentsillustrated therein, the typical process includes sample preparation, abinding reaction, ligation, ligase inactivation using a protease,protease inactivation (e.g., using heat), followed by real-time PCR. Tocarry out the PCR step, a portion of the reaction mixture containing theinactivated ligase and protease is transferred to the PCR plate, and the“PCR mix” (e.g., containing primers, dNTPs, polymerase, and the like)added thereto.

As shown in FIG. 3B, the improved process may eliminate the use of aprotease and dilution of the reaction mixture prior to PCR. As showntherein, the ligase may be inactivated using heat, and the resultantreaction mixture placed directly into the qPCR assay. Thus, someembodiments of the improved PLA work flow uses entire binding reactionproducts in the real-time PCR well. This provides a simplified work-flowand reduced dilution of the reaction mixture. As a result, in somepreferred embodiments of the improved PLA processes, the PCR reactionmixture contains a higher concentration of the ligated product (e.g.,the target nucleic acid).

In order to reduce non-binding probe ligation, the probe concentrationmay be reduced. The splint (e.g., connector) oligo length andconcentration may also be reduced to minimize chance of solutionhybridization promoted by non-antigen-binding ligation (e.g., connectoroligonucleotides of at least 14 bases in length (e.g., 9 basesoverlapping a first oligo probe and 5 bases overlapping a second oligoprobe (9+5)) vs. connector oligonucleotides of at least 18 bases inlength (e.g., 9 bases overlapping a first oligo probe and 9 basesoverlapping a second oligo probe (9+9)). In such embodiments, a smallfootprint ligase (SFL) may be used. As described herein, a SFL mayligate oligonucleotides having a connector oligo length of as short as 3bases of hybridized DNA adjacent to 5′-phosphate hybridized DNA. Forcombining the ligation and PCR reaction into one step, in someembodiments, ATP (cofactor for the ligase) can be optionally omittedfrom the reaction mixture. In order to maintain the ligase function, inother embodiments, the SFL may be pre-enriched with ATP prior to itspurification and use.

In some embodiments of the improved PLA processes splint oligos can beused that are either considered to be symmetrical splints orasymmetrical splints depending on the number of nucleotides thathybridize to each of the two oligo probes it is connecting. FIG. 4A andFIG. 4B diagrams asymmetrical and symmetrical splint types for use inthe improved PLA processes as described herein. Asymmetrical splints (or“connectors”) span across the two separate oligo probes (e.g., probeoligo A and B) with one of the ends of the splint (e.g., either the 3′end or the 5′ end) having more nucleotides that hybridize to one of theprobe oligos than the other end of the splint has nucleotides thathybridize to the alternative probe oligo (FIG. 4A). Symmetrical splintsspan across the two separate oligo probes (e.g., probe oligo A and B)with both ends of the splint (e.g., the 3′ end and the 5′ end) havingequal number of nucleotides that hybridize to each of the two probeoligos (FIG. 4B).

Both asymmetrical and symmetrical splints can have any number ofintervening nucleotides between each of its 3′ and 5′ ends thathybridize to the separate probe oligos. Alternatively, there may be nointervening nucleotides between each of the 3′ and 5′ ends thathybridize to the probe oligos.

Example 3

FIG. 5 provides a comparison between results obtained using exemplaryembodiments of a typical process (TaqMan® Protein Assay Open Kit fromLife Technologies, Inc.; “PLA1”) and an improved process (using methodsdisclosed herein; “PLA2”). Both assays were set up to target CSTB inNTera2 cell lysate. The binding reaction was identical for both PLA1 andPLA2 using the manufacturer's (Life Technologies, Inc.) recommendedreagents and protocol in 4 μl volume binding reactions. After thebinding reaction, PLA1 proceeded following the manufacturer's protocoland reagents. The PLA2 reaction was combined with 16 μl of ligation-PCRreaction mix. The ligation-PCR reaction mix consists of 10 μl theTaqMan® Protein Assay Fast Master Mix (Life Technologies, Inc.), 1 μlUniversal PCR Assay and the connector oligonucleotide 9+5 and the SFLligase, and 5 μl di-water. The ligation reaction was allowed to proceedfor 10 minutes. The ligated product was then placed in a real-time PCRinstrument (Step1Plus) and utilized according to the manufacturer'sinstructions.

As described above, in some embodiments, the improved process (PLA2)carries more target molecules into the PCR step (e.g., resulting in moreamplicons being generated). The improvement provided thereby is shown inFIG. 5. As shown therein, the dCT of the improved process (PLA2) is muchimproved as compared to the typical process (PLA1). In this exemplaryembodiment, the improved process provides at least about a one- tothree-fold dCt improvement over the typical process.

The improved process also provides improved assay sensitivity. As shownby the exemplary embodiment in FIG. 6, the improved process providesabout a two- to ten-fold increase in sensitivity over the typicalprocess (FIG. 6). Sensitivity was calculated as the relativequantification (RQ) fold change using the results from the typicalprocess as the calibrator. The dCt of the improved process wascalculated as fold improvement over the typical process. Since the RQ iscalculated from the dCt threshold of 2, and the fold-change is thereforindicative of the improvement in sensitivity. The data show that thesensitivity of the assay was improved by at least 2-fold, as determinedusing five different targets (GFP, hCSTB, hICAM1, hLIN28, and hOCT3/4).The GFP data was generated using the typical (e.g., PLA1) and improved(e.g., PLA2) processes as described above using a cell lysate into whichrGFP was added (e.g., a “spiked-in” cell lysate) and a GFP probe used.

Example 4

In this example, two different splint lengths were tested at varyingconcentrations.

PLA experiments were carried out using typical PLA conditions (“TaqMan®Protein Assay Open Kit from Life Technologies, Inc.) according to themanufacturers instructions, using a T4 ligase, except that splintconcentrations were varied within the range of 3.1 nM to 1000 nM.Splints were also designed to have a two different splint lengths of 18(9+9; “99”) or 16 (8+8; “88”). Cystatin B (CSTB) assay probes (from“TaqMan® Protein Expression Assay Kit (Human CSTB); Life Technologies,Inc.) were used to detect either 1000 pM or 0 pM (no protein control;“NPC”) of recombinant CSTB protein in buffer. Ct values were plotted foreach splint concentration and delta Ct values (NPC Ct values minus CSTBCt values) and were plotted for each concentration used.

As shown in FIG. 7, a reduction in delta Ct was observed for the 99splint at a low concentration of 3.1 nM as compared to higherconcentrations used. There was also a delta Ct observed for the 88splint at a concentration of 25 nM compared to higher concentrations.Collectively, these data demonstrate that ligated products are reducedwhen the splint length is decreased when the T4 ligase is used.

Example 5

In this example, five different splint lengths were tested using asingle concentration.

PLA experiments were carried out using similar methods as described inExample 4, except that SF ligase instead of T4 ligase was used. Briefly,splints were designed to have a different splint lengths of 12 (3+9), 13(4+9), 14 (5+9 or 7+7), 17 (8+9), or 18 (9+9). The concentration usedfor each of these splints was 100 nM. Raji lysate (Protein ExpressionLysate Control Kit from Life Technologies, Inc.) was prepared at 500cells/reaction or 0 cells/reactiont (“NPC”) and CSTB assay probes (fromTaqMan® Protein Expression Assay Kit (Human CSTB); Life Technologies,Inc.) were used according to the manufacturer's instructions. Ct valueswere plotted for each splint type and delta Ct values (NPC Ct valuesminus 500 cell input Ct values) and were plotted for each.

As shown in FIG. 8, increasing dCT was observed for splints of 12nucleotides in length up to 14 nucleotides in length (including bothasymmetrical and symmetrical splint types). This demonstrates that SFligase is capable of ligating both asymmetric and symmetric splints ofboth shorter and longer lengths.

Example 6

In this example, T4 ligase was compared to two different SF ligases(e.g., SF and DLxD).

PLA experiments were carried out using similar methods as described inExample 5, using the indicated ligases and splints of varying length, asindicated. Briefly, splints were designed to have a two different splintlengths of 14 (5+9; “95”) or 18 (9+9; “99”). The concentration used foreach of these splints was 100 nM. Raji lysate (Protein Expression LysateControl Kit from Life Technologies, Inc.) was prepared at 500cells/reaction or 0 cells/reactiont (“NPC”) and CSTB assay probes (fromTaqMan® Protein Expression Assay Kit (Human CSTB); Life Technologies,Inc.) were used according to the manufacturer's instructions. Ct valueswere plotted for each ligase and splint type and delta Ct values (NPC Ctvalues minus 500 cell input Ct values) and were plotted for each.

As shown in FIG. 9, the T4 ligase resulted no noticeable dCt using the5+9 splint. However, both SF ligases, SF and DLxD, were capable ofligating the target DNA using shorter splint types. In this experiment,SF used with the 5+9 splint resulted in the highest dCt.

The improved processes described herein, and exemplified throughout theExamples above, provide faster times from process start to results(fast), reduce hands-on time (simpler and cheaper), reduce labplasticware usage (cheaper and greener), and increased signals andsensitivities. These improved processes provide simplified work flow bycombining ligation and PCR steps, reduced dilution factor from bindingto ligation step, reduced binding probe concentration to enable reduceddilution factor, use of shorter connector oligo to control backgroundsignal, use lower connector oligo concentration to control backgroundsignal, use of SF ligase to enable use of shorter connector oligolength, ATP enriched SF ligase purification scheme to omit ATP inligation-PCR step, and enabling use of the entire reaction volume toimprove the PLA signal and sensitivity.

While certain embodiments have been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the following claims.

What is claimed is:
 1. A method for ligating at least twooligonucleotides to produce a ligated oligonucleotide and amplifying theligated oligonucleotide, wherein ligation and amplification occur in asingle reaction mixture.
 2. The method of claim 1 whereby a ligand isdetected in a test sample, the method comprising the steps of, incombination: a) contacting a target protein or analyte with at least afirst and second probe, each probe having binding specificity for theprotein or analyte, and being adjoined to at least one type ofoligonucleotide; b) ligating the oligonucleotides on the first andsecond probes to one another using a ligase to produce a target nucleicacid and amplifying the target nucleic acid; and, c) detecting theamplified target nucleic acid.
 3. The method of claim 1 or 2, wherein aportion of at least one of said probes is an antibody.
 4. The method ofclaim 3, wherein a portion of each of said first and second probes areantibodies.
 5. The method of any one of claims 1-4, wherein at least oneof said oligonucleotides comprises at least three nucleotides.
 6. Themethod of any one of claims 1-5, wherein at least one of saidoligonucleotides consists of three nucleotides.
 7. The method of any oneof claims 1-6, wherein said oligonucleotides are ligated using a smallfootprint ligase (SFL).
 8. The method of claim 7, wherein said smallfootprint ligase is contacted with adenosine triphosphate prior to use.9. The method of any one of claims 1-8, wherein said ligatedoligonucleotide is amplified using the polymerase chain reaction (PCR).10. The method claim 9, wherein said amplified ligated oligonucleotideis detected using quantitative PCR (qPCR).
 11. The method of any one ofclaims 1-10, wherein a ligase is used for ligation, and said ligase isinactivated prior to amplification.
 12. The method of claim 11, whereinsaid ligase is inactivated by heat.
 13. The method of claim 11 or 12,wherein said ligase is selected from the group consisting of a nucleicacid ligase, oligonucleotide ligase, DNA ligase, T3 DNA ligase, T4 DNAligase, T5 DNA ligase, T7 DNA ligase, vaccinia virus DNA ligase, E. coliDNA ligase, mammalian DNA ligase I, mammalian DNA ligase II, mammalianDNA ligase III, Tth DNA ligase, KOD DNA ligase, a thermostable DNAligase, RNA ligase, T4 RNA ligase, bacteriophage RB69 RNA ligase,Autographa californica nuclearpolyhedrosis virus RNA ligase, athermophilic RNA ligase, bacteriophage RM378 RNA ligase, bacteriophageTS2126 RNA ligase, a small-footprint DNA ligase (SFL), the ligase of SEQID NO.: 77, the ligase of SEQ ID NO.: 78, the ligase of SEQ ID NO.: 79,the ligase of SEQ ID NO.: 80, the ligase of SEQ ID NO.: 81, the ligaseof SEQ ID NO.: 82, a derivative thereof, a fragment thereof, andcombinations thereof.
 14. A method for detecting a target in a samplewherein: a) binding a first and a second probe, each of which bindsspecifically to the target, wherein each of the probes comprises anoligonucleotide portion or tail; b) ligating the first and secondoligonucleotide tails thereby producing a ligated oligonucleotidetemplate; and, c) performing a polymerase chain reaction (PCR) of theoligonucleotide template across the first and second oligonucleotide toquantify the said template.
 15. The method of claim 14, wherein steps band c are performed in the same reaction mixture.
 16. The method ofclaim 14 or 15, wherein a splint oligonucleotide is used to ligate saidfirst and second oligonucleotide tails.
 17. The method of claim 16,wherein the 3′ and 5′ ends (which overlap with said first and secondoligonucleotide tails, respectively) of said splint oligonucleotide aresymmetrical or asymmetrical to one another.
 18. The method of claim 16or 17, wherein said splint oligonucleotide is blocked at its 3′-end. 19.The method of any of claims 16-18, wherein said splint oligonucleotideis at least 6 nucleotides in length.
 20. The method of any of claims16-19, wherein said 3′-end of said splint oligonucleotide overlaps withat least 3 nucleotides of said first oligonucleotide tail and/or said5′-end of said splint oligonucleotide overlaps with at least 3nucleotides of said second oligonucleotide tail.
 21. The method of anyof claims 14-20, wherein the length of said first and secondoligonucleotide tails is at least 3 nucleotides in length.
 22. Themethod of any of claims 14-21, wherein a small footprint ligase (SFL) isused to ligate said first and second oligonucleotide tails.
 23. Themethod of any of claims 14-22, wherein said first and/or second probesfurther comprises an antibody portion specific to said target.
 24. Amethod for detecting a target in sample comprising: a) binding a firstand a second probe, wherein each probe binds specifically to the target,each of said probes comprise an oligonucleotide portion or tail; b)ligating the oligonucleotides to produce a ligated oligonucleotidetemplate and amplifying the template by PCR in a single step to producean amplified template; and, c) quantitating the amplified template 25.The method of claim 24, wherein the probe further comprises an antibody.26. The method of claim 24 or 25, wherein said ligase is selected fromthe group consisting of a nucleic acid ligase, oligonucleotide ligase,DNA ligase, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7 DNA ligase,vaccinia virus DNA ligase, E. coli DNA ligase, mammalian DNA ligase I,mammalian DNA ligase II, mammalian DNA ligase III, Tth DNA ligase, KODDNA ligase, a thermostable DNA ligase, RNA ligase, T4 RNA ligase,bacteriophage RB69 RNA ligase, Autographa californicanuclearpolyhedrosis virus RNA ligase, a thermophilic RNA ligase,bacteriophage RM378 RNA ligase, bacteriophage TS2126 RNA ligase, asmall-footprint DNA ligase (SFL), the ligase of SEQ ID NO.: 77, theligase of SEQ ID NO.: 78, the ligase of SEQ ID NO.: 79, the ligase ofSEQ ID NO.: 80, the ligase of SEQ ID NO.: 81, the ligase of SEQ ID NO.:82, a derivative thereof, a fragment thereof, and combinations thereof.27. The method of claim 26, wherein said ligase is a T4 DNA ligase. 28.The method of claim 26, wherein the ligase is a small-footprint DNAligase (SFL).
 29. The method of any one of claims 24-27, wherein saidoligonucleotides are ligated using a splint oligonucleotide.
 30. Themethod of claim 28, wherein said splint oligonucleotide comprises a 3′end of four to nine bases in length and a 5′ end of four to nine basesin length.
 31. The method of claim 28 or 29, wherein said 3′ and 5′ ends(which overlap with said first and second tails, respectively) of saidsplint oligonucleotide are symmetrical or asymmetrical to one another.32. The method of claim 28-30, wherein said splint oligonucleotide isblocked at its 3′-end.
 33. The method of any one of claims 23-31,wherein the ligase is pre-enriched using ATP.
 34. The method of any oneof claims 23-32, wherein said ligase is inactivated after ligation usinga protease or heat.
 35. The method of any one of claims 23-33, whereinsaid template is quantified using real-time PCR.
 36. The method of claim34, wherein said real-time PCR assay is a TaqMan® assay.
 37. The methodof claim 1, 14, or 24, wherein said method provides at least about a oneto three-fold dCt improvement over a typical PLA method or process. 38.The method of claim 1, 14, or 24, wherein said method provides about atwo- to ten-fold improvement/increase in sensitivity over the typicalprocess.
 39. The method of claim 36 or 37, wherein said typical methodor process includes the use of a protease and/or dilution of thereaction mixture prior to PCR and said improved method of claim 1, 14,or 24 does not.
 40. The method of claim 2, wherein said oligonucleotideson said first and second probes, are at least partially complementary toone another